Cell-based fluorescence resonance energy transfer (fret) assays for clostridial toxins

ABSTRACT

The present invention provides a method of determining clostridial toxin activity by (a) contacting with a sample a cell containing a clostridial toxin substrate that includes a donor fluorophore; an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and a clostridial toxin recognition sequence containing a cleavage site that intervenes between the donor fluorophore and the acceptor, where resonance energy transfer is exhibited between the donor fluorophore and the acceptor under the appropriate conditions; (b) exciting the donor fluorophore; and (c) determining resonance energy transfer of the contacted cell relative to a control cell, where a difference in resonance energy transfer of the contacted cell as compared to the control cell is indicative of clostridial toxin activity.

This application is a divisional and claims priority pursuant to 35U.S.C. §120 to U.S. patent application Ser. No. 10/261,161, filed Sep.27, 2002, which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to fluorescence resonance energytransfer and protease assays and, more specifically, to cell-basedmethods for assaying for clostridial toxin activity.

2. Background Information

The neuroparalytic syndrome of tetanus and the rare but potentiallyfatal disease, botulism, are caused by neurotoxins produced by bacteriaof the genus Clostridium. These clostridial neurotoxins are highlypotent and specific poisons of neural cells, with the human lethal doseof the botulinum toxins on the order of nanograms. Thus, the presence ofeven minute levels of botulinum toxins in foodstuffs represents a publichealth hazard that must be avoided through rigorous testing.

However, in spite of their potentially deleterious effects, lowcontrolled doses of botulinum neurotoxins have been successfully used astherapeutics and for some cosmetic applications. In particular,botulinum toxins have been used in the therapeutic management of avariety of focal and segmental dystonias, strabismus, and otherconditions in which a reversible depression of a cholinergic nerveterminal activity is desired. Established therapeutic uses of botulinumneurotoxins in humans include, without limitation, blepharospasm,hemifacial spasm, laringeal dysphonia, focal hyperhidrosis,hypersalivation, oromandibular dystonia, cervical dystonia, torticollis,strabismus, limbs dystonia, occupational cramps and myokymia (Rossettoet al., Toxicon 39:27-41 (2001)). As an example, intramuscular injectionof spastic tissue with small quantities of botulinum neurotoxin A hasbeen used effectively to treat spasticity due to brain injury, spinalcord injury, stroke, multiple sclerosis and cerebral palsy. Additionalpossible clinical uses of clostridial neurotoxins currently are beinginvestigated.

Given the potential danger associated with small quantities of botulinumtoxins in foodstuffs and the need to prepare accurate pharmaceuticalformulations, assays for botulinum neurotoxins presently are employed inthe food and pharmaceutical industries. The food industry requiresassays for the botulinum neurotoxins to validate new food packagingmethods and to ensure food safety. The growing clinical use of thebotulinum toxins necessitates accurate assays for botulinum neurotoxinactivity for product formulation as well as quality control. In bothindustries, a mouse lethality test currently is the only acceptableassay for botulinum neurotoxin activity.

Unfortunately, the mouse lethality assay suffers from several drawbacks:cost due to the large numbers of laboratory animals required; lack ofspecificity; the potential for inaccuracy unless large animal groups areused; and the necessary sacrifice of animal life. Thus, there is a needfor a new method that can complement and reduce the need for the mouselethality assay. In addition to measuring toxin proteolytic activity,such a surrogate method also should require cellular uptake of the toxinand delivery of the toxin light chain into the cell cytosol. The presentinvention satisfies this need by providing novel cell-based assays forclostridial toxin activity and also provides related advantages.

SUMMARY OF THE INVENTION

The present invention provides a substrate composition that includes adelivery agent and a clostridial toxin substrate containing a donorfluorophore; an acceptor having an absorbance spectrum overlapping theemission spectrum of the donor fluorophore; and a clostridial toxinrecognition sequence containing a cleavage site that intervenes betweenthe donor fluorophore and the acceptor, where resonance energy transferis exhibited between the donor fluorophore and the acceptor under theappropriate conditions. In a substrate composition of the invention, thedelivery agent can be, for example, covalently linked to the clostridialtoxin substrate and further can be, for example, a protein, peptide orpeptidomimetic. In one embodiment, the substrate composition is achimeric protein, peptide or peptidomimetic in which the delivery agentis operatively fused to the clostridial toxin substrate. Such a chimericsubstrate composition can be, for example, a peptide or peptidomimetichaving a length of at most 50 or 100 residues.

A variety of delivery agents can be covalently linked to a clostridialtoxin substrate in a substrate composition of the invention including,without limitation, an antennapedia protein or active fragment thereof,such as an active fragment having the amino acid sequenceRQIKIWFQNRRMKWKK (SEQ ID NO: 1); an HIV TAT protein or active fragmentthereof, such as an active fragment having the amino acid sequenceYGRKKRRQRRR (SEQ ID NO: 2); and a herpes simplex virus VP22 protein oractive fragment thereof, such as a herpes simplex virus VP22 proteinhaving the amino acid sequence SEQ ID NO: 3, or active fragment thereof.

The invention also provides a substrate composition in which thedelivery agent is non-covalently associated with the clostridial toxinsubstrate. Exemplary delivery agents that can be non-covalentlyassociated with a clostridial toxin substrate include, withoutlimitation, Chariot™ and MPG peptides.

A variety of clostridial toxin substrates are useful in the substratecompositions of the invention. Such a clostridial toxin substrate canbe, for example, a botulinum toxin substrate containing a botulinumtoxin recognition sequence or a tetanus toxin substrate containing atetanus toxin recognition sequence. In one embodiment, the inventionprovides a substrate composition containing, in part, a BoNT/A substratethat includes a BoNT/A recognition sequence. Such a BoNT/A substrate caninclude, for example, at least six consecutive residues of SNAP-25, thesix consecutive residues containing Gln-Arg, or a peptidomimeticthereof. In another embodiment, the invention provides a substratecomposition that includes a BoNT/B substrate containing a BoNT/Brecognition sequence. BoNT/B substrates useful in the substratecompositions of the invention include, without limitation, those havingat least six consecutive residues of VAMP, the six consecutive residuescontaining Gln-Phe, or a peptidomimetic thereof. In a furtherembodiment, the invention provides a BoNT/C1 substrate containing aBoNT/C1 recognition sequence; BoNT/C1 substrates useful in the inventionencompass those having at least six consecutive residues of syntaxin,the six consecutive residues containing Lys-Ala, or a peptidomimeticthereof, and those including at least six consecutive residues ofSNAP-25, the six consecutive residues containing Arg-Ala, or apeptidomimetic thereof. In another embodiment, the invention provides aBoNT/D substrate containing a BoNT/D recognition sequence. A variety ofBoNT/D substrates are useful in the substrate compositions of theinvention including, yet not limited to, BoNT/D substrates containing atleast six consecutive residues of VAMP, the six consecutive residuescontaining Lys-Leu, or a peptidomimetic thereof.

In a further embodiment, the invention provides a substrate compositionthat includes, in part, a BoNT/E substrate containing a BoNT/Erecognition sequence. Such a BoNT/E substrate can have, for example, atleast six consecutive residues of SNAP-25, the six consecutive residuescontaining Arg-Ile, or a peptidomimetic thereof. In an additionalembodiment, there is provided herein a substrate composition whichincludes a BoNT/F substrate containing a BoNT/F recognition sequence.BoNT/F substrates useful in the compositions of the invention can have,for example, at least six consecutive residues of VAMP, the sixconsecutive residues containing Gln-Lys, or a peptidomimetic thereof. Instill a further embodiment, the invention provides a substratecomposition that contains, in part, a BoNT/G substrate having a BoNT/Grecognition sequence. Useful BoNT/G substrates include, yet are notlimited to, those having at least six consecutive residues of VAMP, thesix consecutive residues containing Ala-Ala, or a peptidomimeticthereof. In a further embodiment, the present invention provides asubstrate composition that includes, in part, a TeNT substratecontaining a TeNT recognition sequence. A variety of TeNT substrates areuseful in the invention including those having at least six consecutiveresidues of VAMP, the six consecutive residues containing Gln-Phe, or apeptidomimetic thereof.

A variety of donor fluorophores and acceptors are useful in thesubstrate compositions of the invention. As non-limiting examples, donorfluorophores useful in the invention include Alexa Fluor®-488, DABCYLand BODIPY®. Acceptors useful in the invention include non-fluorescentacceptors as well as acceptor fluorophores; in one embodiment, theacceptor is an acceptor fluorophore having a fluorescence lifetime of atleast 1 microsecond. In other embodiments, the invention provides asubstrate composition in which the acceptor is EDANS, QSY-7®, ortetramethylrhodamine.

Further provided herein is a cell containing a clostridial toxinsubstrate that includes a donor fluorophore; an acceptor having anabsorbance spectrum overlapping the emission spectrum of the donorfluorophore; and a clostridial toxin recognition sequence containing acleavage site that intervenes between the donor fluorophore and theacceptor, where resonance energy transfer is exhibited between the donorfluorophore and the acceptor under the appropriate conditions. In oneembodiment, the cell is a transfected cell. In another embodiment, thecell is a stably transfected cell. A variety of cells are useful in theinvention including, without limitation, primary cells; establishedcells; human cells; neuronal cells such as primary neurons, establishedneurons and human neurons; and non-neuronal cells such as pancreaticacinar cells. Neurons useful in the invention include central nervoussystem (CNS) neurons and peripheral neurons; as non-limiting examples, aneuron useful in the invention can be a neuroblastoma, spinal cordneuron, dorsal root ganglion neuron, cerebral cortex neuron, cerebellarneuron, hippocampal neuron or motor neuron.

Further provided herein is a cell which includes a nucleic acid moleculeencoding a clostridial toxin substrate that includes a donorfluorophore; an acceptor having an absorbance spectrum overlapping theemission spectrum of the donor fluorophore; and a clostridial toxinrecognition sequence containing a cleavage site that intervenes betweenthe donor fluorophore and the acceptor, where resonance energy transferis exhibited between the donor fluorophore and the acceptor under theappropriate conditions.

Any of a variety of cells can be useful, including, but not limited to,human cells, neuronal cells and non-neuronal cells. Such a cell can beprepared, for example, by stable transfection of a nucleic acid moleculeencoding a clostridial toxin substrate. The nucleic acid moleculeencoding a clostridial toxin substrate can be linked, for example, to aregulatory element such as a constitutive regulatory element orinducible regulatory element. A variety of inducible regulatory elementsare useful in the invention, including, without limitation, tetracyclineregulated regulatory elements and ecdysone inducible regulatoryelements. Genetically encoded donor fluorophores and acceptors useful inthe invention include green fluorescence protein (GFP) and othersdisclosed herein below and known in the art.

The present invention also provides a method of determining clostridialtoxin activity by (a) contacting with a sample a cell containing aclostridial toxin substrate that includes a donor fluorophore; anacceptor having an absorbance spectrum overlapping the emission spectrumof the donor fluorophore; and a clostridial toxin recognition sequencecontaining a cleavage site that intervenes between the donor fluorophoreand the acceptor, where resonance energy transfer is exhibited betweenthe donor fluorophore and the acceptor under the appropriate conditions;(b) exciting the donor fluorophore; and (c) determining resonance energytransfer of the contacted cell relative to a control cell, where adifference in resonance energy transfer of the contacted cell ascompared to the control cell is indicative of clostridial toxinactivity.

A clostridial toxin substrate useful in a method of the invention can bea botulinum toxin substrate of any serotype or a tetanus toxinsubstrate.

Thus, a method of the invention can be practiced, for example, with aBoNT/A substrate containing a BoNT/A recognition sequence; a BoNT/Bsubstrate containing a BoNT/B recognition sequence; a BoNT/C1 substratecontaining a BoNT/C1 recognition sequence; a BoNT/D substrate containinga BoNT/D recognition sequence; a BoNT/E substrate containing a BoNT/Erecognition sequence; a BoNT/F substrate containing a BoNT/F recognitionsequence; a BoNT/G substrate containing a BoNT/G recognition sequence;or a TeNT toxin substrate containing a TeNT recognition sequence.

A variety of samples can be assayed for clostridial toxin activityaccording to a method of the invention. Such samples include, withoutlimitation, crude cell lysates; isolated clostridial toxins; formulatedclostridial toxin products such as BOTOX; and foodstuffs.

A variety of means can be used to determine resonance energy transfer ina method of the invention. In one embodiment, a method of the inventionincludes the step of detecting donor fluorescence intensity of thecontacted cell, where increased donor fluorescence intensity of thecontacted cell as compared to the control cell is indicative ofclostridial toxin activity. In another embodiment, a method of theinvention includes the step of detecting acceptor fluorescence intensityof the contacted cell, where decreased acceptor fluorescence intensityof the contacted cell as compared to the control cell is indicative ofclostridial toxin activity. In a further embodiment, a method of theinvention includes the step of detecting an acceptor emission maximumand a donor fluorophore emission maximum of the contacted cell, where ashift in emission maxima from near the acceptor emission maximum to nearthe donor fluorophore emission maximum is indicative of clostridialtoxin activity. In yet a further embodiment, a method of the inventionincludes the step of detecting the ratio of fluorescence amplitudes nearan acceptor emission maximum to the fluorescence amplitudes near a donorfluorophore emission maximum of the contacted cell, where a decreasedratio in the contacted cell as compared to the control cell isindicative of clostridial toxin activity. In still another embodiment, amethod of the invention includes the step of detecting the excited statelifetime of the donor fluorophore in the contacted cell, where anincreased donor fluorophore excited state lifetime in the contacted cellas compared to the control cell is indicative of clostridial toxinactivity. If desired, the step of determining resonance energy transfercan be repeated at one or more later time intervals. In addition, theconditions suitable for clostridial toxin activity can be selected, ifdesired, such that the assay is linear.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the deduced structure and postulatedmechanism of activation of clostridial neurotoxins. Toxins can beproduced as an inactive single polypeptide chain of 150 kDa, composed ofthree 50 kDa domains connected by loops. Selective proteolytic cleavageactivates the toxins by generating two disulfide-linked chains: the Lchain of 50 kDa and the H chain of 100 kDa, which is made up of twodomains denoted H_(N) and H_(C). The three domains play distinct roles:the C-terminal domain of the heavy chain (H_(C)) functions in cellbinding while the N-terminal domain of the heavy chain (H_(N)) permitstranslocation from endosome to cell cytoplasm. Following reduction ofthe disulfide linkage inside the cell, the zinc-endopeptidase activityof the L chain is liberated.

FIG. 2 shows a schematic of the four steps required for tetanus andbotulinum toxin activity in central and peripheral neurons.

FIG. 3 shows the subcellular localization at the plasma membrane andsites of cleavage of SNAP-25, VAMP and syntaxin. VAMP is bound tosynaptic vesicle membrane, whereas SNAP-25 and syntaxin are bound to thetarget plasma membrane. BoNT/A and /E cleave SNAP-25 close to thecarboxy-terminus, releasing nine or 26 residues, respectively. BoNT/B,/D, /F, /G and TeNT act on the conserved central portion of VAMP(dotted) and release the amino-terminal portion of VAMP into thecytosol. BoNT/C1 cleaves SNAP-25 close to the carboxy-terminus as wellas cleaving syntaxin at a single site near the cytosolic membranesurface. The action of BoNT/B, /C1, /D, /F, /G and TeNT results inrelease of a large portion of the cytosolic domain of VAMP or syntaxin,while only a small portion of SNAP-25 is released by selectiveproteolysis by BoNT/A, /C1 or /E.

FIG. 4 shows the neurotoxin recognition motif of VAMP, SNAP-25 andsyntaxin. (A) Hatched boxes indicate the presence and positions of amotif common to the three targets of clostridial neurotoxins. (B) Therecognition motif is composed of hydrophobic residues (“h”); negativelycharged Asp or Glu residues (“−”) and polar residues (“p”); “x”represents any amino acid. The motif is included in regions of VAMP,SNAP-25 and syntaxin predicted to adopt an α-helical conformation. (C) Atop view of the motif in an α-helical conformation is shown. Negativelycharged residues align on one face, while hydrophobic residues align ona second face.

FIG. 5 shows an alignment of various SNAP-25 proteins and their BoNT/E,BoNT/A and BoNT/C1 cleavage sites. Human SNAP-25 (SEQ ID NO: 4; GenBankaccession g4507099; see, also, related human SNAP-25 sequence g2135800);mouse SNAP-25 (SEQ ID NO: 5; GenBank accession G6755588); DrosophilaSNAP-25 (SEQ ID NO: 6; GenBank accession g548941); goldfish SNAP-25 (SEQID NO: 7; GenBank accession g2133923); sea urchin SNAP-25 (SEQ ID NO: 8;GenBank accession g2707818) and chicken SNAP-25 (SEQ ID NO: 9; GenBankaccession g481202) are depicted.

FIG. 6 shows an alignment of various VAMP proteins and their BoNT/F,BoNT/D, BoNT/B, TeNT and BoNT/G cleavage sites. Human VAMP-1 (SEQ ID NO:10; GenBank accession g135093); human VAMP-2 (SEQ ID NO: 11; GenBankaccession g135094); mouse VAMP-2 (SEQ ID NO: 12; GenBank accessiong2501081); bovine VAMP (SEQ ID NO: 13; GenBank accession g89782); frogVAMP (SEQ ID NO: 14; GenBank accession g6094391); and sea urchin VAMP(SEQ ID NO: 15; GenBank accession g5031415) are depicted.

FIG. 7 shows an alignment of various syntaxin proteins and their BoNT/C1cleavage sites. Human syntaxin 1A (SEQ ID NO: 16; GenBank accessiong15079184), human syntaxin 1B2 (SEQ ID NO: 17; GenBank accessiong15072437), mouse syntaxin 1A (SEQ ID NO: 18; GenBank accessiong15011853), Drosophila syntaxin 1A (SEQ ID NO: 19; GenBank accessiong2501095); C. elegans syntaxin A (SEQ ID NO: 20; GenBank accessiong7511662)and sea urchin syntaxin (SEQ ID NO: 21; GenBank accessiong13310402) are depicted.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides in vivo and in vitro cell-based assays fordetermining the presence or absence of an active clostridial toxin in asample or for determining the activity of any clostridial toxin,including botulinum toxins of all serotypes and tetanus toxins. Thenovel substrate compositions, cells and assays of the invention reducethe need for animal toxicity studies, yet serve to analyze multipletoxin functions, namely, binding and cellular uptake of the toxin,translocation into the cell cytosol, and protease activity. These novelcompositions and methods can be used to analyze crude and bulk samplesas well as highly purified dichain toxins or formulated toxin productsand, furthermore, are amenable to automated high-throughput assayformats.

As discussed below, fluorescence resonance energy transfer (FRET) is adistance-dependent interaction between the electronic excited states oftwo molecules in which excitation is transferred from a donorfluorophore to an acceptor without emission of a photon. The process ofenergy transfer results in a reduction (quenching) of fluorescenceintensity and excited state lifetime of the donor fluorophore and, wherethe acceptor is a fluorophore, can produce an increase in the emissionintensity of the acceptor. Upon cleavage of the clostridial toxinsubstrate within a cell of the invention after being contacted with asample containing active toxin, resonance energy transfer is reduced andcan be detected, for example, by increased donor fluorescence emission,decreased acceptor fluorescence emission, or by a shift in the emissionmaxima from near the acceptor emission maxima to near the donor emissionmaxima. If desired, the amount of active clostridial toxin in a samplecan be calculated as a function of the difference in the degree of FRETusing the appropriate standards.

The tetanus and botulinum neurotoxins to which the invention relates areproduced by Clostridia. These toxins cause the neuroparalytic syndromesof tetanus and botulism, with tetanus toxin acting mainly within thecentral nervous system and botulinum toxin acting on the peripheralnervous system. Clostridial neurotoxins share a similar mechanism ofcell intoxication in which the release of neurotransmitters is blocked.In these toxins, which are composed of two disulfide-linked polypeptidechains, the larger subunit is responsible for neurospecific binding andtranslocation of the smaller subunit into the cytoplasm. Upontranslocation and reduction in neurons, the smaller chain displayspeptidase activity specific for protein components involved inneuroexocytosis. The “SNARE” protein targets of clostridial toxins arecommon to exocytosis in a variety of non-neuronal types; in these cells,as in neurons, light chain peptidase activity inhibits exocytosis.

Tetanus neurotoxin and botulinum neurotoxins B, D, F, and G specificallyrecognize VAMP (also known as synaptobrevin), an integral protein of thesynaptic vesicle membrane. VAMP is cleaved at distinct bonds dependingon the neurotoxin. Botulinum A and E neurotoxins recognize and cleavespecifically SNAP-25, a protein of the presynaptic membrane, at twodifferent sites in the carboxy-terminal portion of the protein.Botulinum neurotoxin C cleaves syntaxin, a protein of the nerveplasmalemma, in addition to SNAP-25. The three protein targets of theClostridial neurotoxins are conserved from yeast to humans althoughcleavage sites and toxin susceptibility are not necessarily conserved(see below; see, also, Humeau et al., Biochimie 82:427-446 (2000);Niemann et al., Trends in Cell Biol. 4:179-185 (1994); and Pellizzari etal., Phil. Trans. R. Soc. London 354:259-268 (1999)).

Naturally occurring tetanus and botulinum neurotoxins are produced asinactive polypeptide chains of 150 kDa without a leader sequence. Thesetoxins may be cleaved by bacterial or tissue proteinases at an exposedprotease-sensitive loop, generating active dichain toxin. Naturallyoccurring clostridial toxins contain a single interchain disulfide bondbridging the heavy chain (H, 100 kDa) and light chain (L, 50 kDa); sucha bridge is important for neurotoxicity of toxin added extracellularly(Montecucco and Schiavo, Quarterly Rev. Biophysics 28:423-472 (1995)).

The clostridial toxins appear to be folded into three distinct 50 kDadomains, as shown in FIG. 1, with each domain having a distinctfunctional role. As illustrated in FIG. 2, the cell intoxicationmechanism of the clostridial toxins consists of four distinct steps: (1)binding; (2) internalization; (3) membrane translocation; and (4)enzymatic target modification. The carboxy-terminal part of the heavychain (H_(C)) functions in neurospecific binding, while theamino-terminal portion of the H chain (H_(N)) functions in membranetranslocation. The L chain is responsible for the intracellularcatalytic activity (Montecucco and Schiavo, supra, 1995).

The amino acid sequence of eight human clostridial neurotoxins has beenderived from the corresponding gene (Neimann, “Molecular Biology ofClostridial Neurotoxins” in Sourcebook of Bacterial Protein Toxins Aloufand Freer (Eds.) pp. 303-348 London: Academic Press 1991). The L chainsand H chains are composed of roughly 439 and 843 residues, respectively.Homologous segments are separated by regions of little or no similarity.The most well conserved regions of the L chains are the amino-terminalportion (100 residues) and central region (corresponding to residues 216to 244 of TeNT), as well as the two cysteines forming the interchaindisulfide bond. The 216 to 244 region contains a His-Glu-X—X-His bindingmotif characteristic of zinc-endopeptidases.

The clostridial toxin heavy chains are less well conserved than thelight chains, and the carboxy-terminal portion of H_(C) (correspondingto residues 1140 to 1315 of TeNT) is the most variable. This isconsistent with the involvement of the H_(C) domain in binding to nerveterminals and the fact that different neurotoxins appear to binddifferent receptors.

Comparison of the nucleotide and amino acid sequences of clostridialtoxins indicates that they derive from a common ancestral gene.Spreading of these genes may have been facilitated by the fact that theclostridial neurotoxin genes are located on mobile genetic elements. Asdiscussed further below, sequence variants of the seven botulinum toxinsare known in the art. See, for example, FIGS. 5 to 7 and Humeau et al.,supra, 2000.

As discussed above, natural targets of the clostridial neurotoxinsinclude VAMP, SNAP-25, and syntaxin. VAMP is bound to the synapticvesicle membrane, whereas SNAP-25 and syntaxin are bound to the targetmembrane (see FIG. 3). BoNT/A and BoNT/E cleave SNAP-25 in thecarboxy-terminal region, releasing nine or twenty-six amino acidresidues, respectively, and BoNT/C1 also cleaves SNAP-25 near thecarboxy-terminus. The botulinum serotypes BoNT/B, BoNT/D, BoNT/F andBoNT/G, and tetanus toxin, act on the conserved central portion of VAMP,and release the amino-terminal portion of VAMP into the cytosol. BoNT/C1cleaves syntaxin at a single site near the cytosolic membrane surface.Thus, BoNT/B, BoNT/C1, BoNT/D, BoNT/F, BoNT/G or TeNT proteolysisresults in release of a large portion of the cytosolic domain of VAMP orsyntaxin, while only a small portion of SNAP-25 is released by BoNT/A,BoNT/C1 or BoNT/E cleavage (Montecucco and Schiavo, supra, 1995).

Naturally occurring SNAP-25, a protein of about 206 residues lacking atransmembrane segment, is associated with the cytosolic surface of thenerve plasmalemma (FIG. 3; see, also, Hodel et al., Int. J. Biochemistryand Cell Biology 30:1069-1073 (1998)). In addition to homologs highlyconserved from Drosophila to mammals, SNAP-25-related proteins also havebeen cloned from yeast. SNAP-25 is required for axonal growth duringdevelopment and may be required for nerve terminal plasticity in themature nervous system. In humans, two isoforms are differentiallyexpressed during development; isoform a is constitutively expressedduring fetal development, while isoform b appears at birth andpredominates in adult life. SNAP-25 analogues such as SNAP-23 also areexpressed outside the nervous system, for example, in pancreatic cells.

Naturally occurring VAMP is a protein of about 120 residues, with theexact length depending on the species and isotype. As shown in FIG. 3,VAMP contains a short carboxy-terminal segment inside the vesicle lumenwhile most of the molecule is exposed to the cytosol. The proline-richamino-terminal thirty residues are divergent among species and isoformswhile the central portion of VAMP (residues 30 to 96), which is rich incharged and hydrophilic residues and includes known cleavage sites, ishighly conserved. VAMP colocalizes with synaptophysin on the synapticvesicle membrane.

A variety of species homologs of VAMP are known in the art includinghuman, rat, bovine, Torpedo, Drosophila, yeast, squid and Aplysiahomologs. In addition, multiple isoforms of VAMP have been identifiedincluding VAMP-1, VAMP-2 and cellubrevin, and insensitive forms havebeen identified in non-neuronal cells. VAMP appears to be present in allvertebrate tissues although the distribution of VAMP-1 and VAMP-2 variesin different cell types. Chicken and rat VAMP-1 are not cleaved by TeNTor BoNT/B. These VAMP-1 homologs have a valine in place of the glutaminepresent in human and mouse VAMP-1 at the TeNT or BoNT/B cleavage site.The substitution does not effect BoNT/D, /F or /G, which cleave bothVAMP-1 and VAMP-2 with similar rates.

Syntaxin is located on the cytosolic surface of the nerve plasmalemmaand is membrane-anchored via a carboxy-terminal segment, with most ofthe protein exposed to the cytosol. Syntaxin colocalizes with calciumchannels at the active zones of the presynaptic membrane, whereneurotransmitter release takes place. In addition, syntaxin interactswith synaptotagmin, a protein of the SSV membrane, that forms afunctional bridge between the plasmalemma and the vesicles. A variety ofsyntaxin isoforms have been identified. Two isoforms of slightlydifferent length (285 and 288 residues) have been identified in nervecells (isoforms 1A and 1B), with isoforms 2, 3, 4 and 5 expressed inother tissues. The different isoforms have varying sensitivities toBoNT/C1, with the 1A, 1B, 2 and 3 syntaxin isoforms cleaved by thistoxin.

The present invention provides a substrate composition that includes adelivery agent and a clostridial toxin substrate containing a donorfluorophore; an acceptor having an absorbance spectrum overlapping theemission spectrum of the donor fluorophore; and a clostridial toxinrecognition sequence containing a cleavage site that intervenes betweenthe donor fluorophore and the acceptor, where resonance energy transferis exhibited between the donor fluorophore and the acceptor under theappropriate conditions. In a substrate composition of the invention, thedelivery agent can be, for example, covalently linked to the clostridialtoxin substrate and further can be, for example, a protein, peptide orpeptidomimetic. In one embodiment, the substrate composition is achimeric protein, peptide or peptidomimetic in which the delivery agentis operatively fused to the clostridial toxin substrate. Such a chimericsubstrate composition can be, for example, a peptide or peptidomimetichaving a length of at most 50 or 100 residues.

A variety of delivery agents can be covalently linked to a clostridialtoxin substrate in a substrate composition of the invention including,without limitation, an antennapedia protein or active fragment thereof,such as an active fragment having the amino acid sequenceRQIKIWFQNRRMKWKK (SEQ ID NO: 1); an HIV TAT protein or active fragmentthereof, such as an active fragment having the amino acid sequenceYGRKKRRQRRR (SEQ ID NO: 2); or a herpes simplex virus VP22 protein oractive fragment thereof, such as a herpes simplex virus VP22 proteinhaving the amino acid sequence SEQ ID NO: 3, or active fragment thereof.

The invention also provides substrate compositions in which the deliveryagent is non-covalently associated with the clostridial toxin substrate.Exemplary delivery agents that can be non-covalently associated with aclostridial toxin substrate include, without limitation, Chariot™ andMPG peptides.

A variety of donor fluorophores and acceptors are useful in thesubstrate compositions of the invention. As non-limiting examples, donorfluorophores useful in the invention include Alexa Fluor®-488, DABCYLand BODIPY®. Acceptors useful in the invention include non-fluorescentacceptors as well as acceptor fluorophores; in one embodiment, theacceptor is an acceptor fluorophore having a fluorescence lifetime of atleast 1 microsecond. In other embodiments, the invention provides asubstrate composition in which the acceptor is tetramethylrhodamine,EDANS or QSY-7®. Exemplary donor fluorophore-acceptor pairs useful in asubstrate composition of the invention include, without limitation,fluorescein-tetramethylrhodamine, Alexa Fluor® 488-tetramethylrhodamine,DABCYL-EDANS, fluorescein-QSY® 7, and Alexa Fluor® 488-QSY® 7.

A variety of clostridial toxin substrates are useful in the substratecompositions of the invention. Such clostridial toxin substrates includebotulinum toxin substrates containing a botulinum toxin recognitionsequence and tetanus toxin substrates containing a tetanus toxinrecognition sequence. Thus, in one embodiment, the invention provides aBoNT/A substrate composition containing a delivery agent and a BoNT/Asubstrate, where the substrate includes a donor fluorophore; an acceptorhaving an absorbance spectrum overlapping the emission spectrum of thedonor fluorophore; and a BoNT/A recognition sequence that contains acleavage site that intervenes between the donor fluorophore and theacceptor and where, under the appropriate conditions, resonance energytransfer is exhibited between the donor fluorophore and the acceptor.The BoNT/A substrate can include, for example, at least six consecutiveresidues of SNAP-25, the six consecutive residues containing Gln-Arg, ora peptidomimetic thereof. A BoNT/A substrate composition of theinvention can be, for example, a peptide or peptidomimetic having atmost twenty, thirty, forty, fifty or 100 residues and can include any ofa variety of donor fluorophore-acceptor combination such as, withoutlimitation, fluorescein-tetramethylrhodamine, DABCYL-EDANS, and AlexaFluor®-488-QSY-7®.

The present invention also provides a BoNT/B substrate compositioncontaining a delivery agent and a BoNT/B substrate, where the substrateincludes a donor fluorophore; an acceptor having an absorbance spectrumoverlapping the emission spectrum of the donor fluorophore; and a BoNT/Brecognition sequence that contains a cleavage site that intervenesbetween the donor fluorophore and the acceptor and where, under theappropriate conditions, resonance energy transfer is exhibited betweenthe donor fluorophore and the acceptor. A BoNT/B substrate useful in theinvention can include, for example, at least six consecutive residues ofVAMP, the six consecutive residues containing Gln-Phe, or apeptidomimetic thereof. A BoNT/B substrate composition of the inventioncan be, for example, a peptide or peptidomimetic having at most twenty,thirty, forty, fifty or 100 residues. It is understood that a variety ofdonor fluorophore-acceptor combinations are useful in a BoNT/B substratecomposition of the invention; such donor fluorophore-acceptor pairsinclude, but are not limited to, fluorescein-tetramethylrhodamine,DABCYL-EDANS, and Alexa Fluor®-488-QSY-7®.

Further provided herein is a BoNT/C1 substrate composition containing adelivery agent and a BoNT/C1 substrate, where the substrate includes adonor fluorophore; an acceptor having an absorbance spectrum overlappingthe emission spectrum of the donor fluorophore; and a BoNT/C1recognition sequence that contains a cleavage site that intervenesbetween the donor fluorophore and the acceptor and where, under theappropriate conditions, resonance energy transfer is exhibited betweenthe donor fluorophore and the acceptor. BoNT/C1 substrates useful in thesubstrate compositions of the invention encompass those having at leastsix consecutive residues of syntaxin, the six consecutive residuescontaining Lys-Ala, or a peptidomimetic thereof, and those including atleast six consecutive residues of SNAP-25, the six consecutive residuescontaining Arg-Ala, or a peptidomimetic thereof. A BoNT/C1 substratecomposition of the invention can be, for example, a peptide orpeptidomimetic having a variety of lengths, for example, at most twenty,thirty, forty, fifty or 100 residues A variety of donorfluorophore-acceptor combinations are useful in a BoNT/C1 substratecomposition of the invention including, without limitation,fluorescein-tetramethylrhodamine, DABCYL-EDANS, and AlexaFluor®-488-QSY-7®.

The invention also provides a BoNT/D substrate composition containing adelivery agent and a BoNT/D substrate, where the substrate includes adonor fluorophore; an acceptor having an absorbance spectrum overlappingthe emission spectrum of the donor fluorophore; and a BoNT/D recognitionsequence containing a cleavage site that intervenes between the donorfluorophore and the acceptor and where, under the appropriateconditions, resonance energy transfer is exhibited between the donorfluorophore and the acceptor. A variety of BoNT/D substrates are usefulin the substrate compositions of the invention including, yet notlimited to, BoNT/D substrates containing at least six consecutiveresidues of VAMP, the six consecutive residues containing Lys-Leu, or apeptidomimetic thereof. As for the other substrate compositionsdiscussed above, a BoNT/D substrate composition can be a peptide orpeptidomimetic having, for example, at most twenty, thirty, forty, fiftyor 100 residues and can include any of a variety of donorfluorophore-acceptor combinations such as, for example,fluorescein-tetramethylrhodamine, DABCYL-EDANS, or AlexaFluor®-488-QSY-7®.

The invention additionally provides a BoNT/E substrate compositioncontaining a delivery agent and a BoNT/E substrate, where the substrateincludes a donor fluorophore; an acceptor having an absorbance spectrumoverlapping the emission spectrum of the donor fluorophore; and a BoNT/Erecognition sequence which contains a cleavage site that intervenesbetween the donor fluorophore and the acceptor and where, under theappropriate conditions, resonance energy transfer is exhibited betweenthe donor fluorophore and the acceptor. A BoNT/E substrate useful in theinvention can have, for example, at least six consecutive residues ofSNAP-25, the six consecutive residues containing Arg-Ile, or apeptidomimetic thereof. A BoNT/E substrate composition of the inventioncan be, for example, a peptide or peptidomimetic having at most twenty,thirty, forty, fifty or 100 residues. Such a substrate compositionfurther can include, for example, a donor fluorophore-acceptorcombination such as fluorescein-tetramethylrhodamine, DABCYL-EDANS orAlexa Fluor® 488-QSY 7®.

Also provided herein is a BoNT/F substrate composition containing adelivery agent and a BoNT/F substrate, where the substrate includes adonor fluorophore; an acceptor having an absorbance spectrum overlappingthe emission spectrum of the donor fluorophore; and a BoNT/F recognitionsequence containing a cleavage site that intervenes between the donorfluorophore and the acceptor and where, under the appropriateconditions, resonance energy transfer is exhibited between the donorfluorophore and the acceptor. BoNT/F substrates useful in the substratecompositions of the invention can have, for example, at least sixconsecutive residues of VAMP, the six consecutive residues containingGln-Lys, or a peptidomimetic thereof. Furthermore, a BoNT/F substratecomposition of the invention can be, for example, a peptide orpeptidomimetic having at most twenty, thirty, forty, fifty or 100residues. One skilled in the art understands that any of a variety offluorophore-acceptor combinations are useful in a BoNT/F substratecomposition; these include, as non-limiting examples,fluorescein-tetramethylrhodamine, DABCYL-EDANS, and AlexaFluor®-488-QSY-7®.

The present invention further provides a BoNT/G substrate compositioncontaining a delivery agent and a BoNT/G substrate, where the substrateincludes a donor fluorophore; an acceptor having an absorbance spectrumoverlapping the emission spectrum of the donor fluorophore; and a BoNT/Grecognition sequence containing a cleavage site that intervenes betweenthe donor fluorophore and the acceptor and where, under the appropriateconditions, resonance energy transfer is exhibited between the donorfluorophore and the acceptor. Useful BoNT/G substrates include, yet arenot limited to, those having at least six consecutive residues of VAMP,the six consecutive residues containing Ala-Ala, or a peptidomimeticthereof. A BoNT/G substrate composition of the invention can be, forexample, a peptide or peptidomimetic having at most twenty, thirty,forty, fifty or 100 residues and can include, for example, a donorfluorophore-acceptor combination such asfluorescein-tetramethylrhodamine, DABCYL-EDANS, or AlexaFluor®-488-QSY-7®.

The invention also provides a TeNT substrate composition containing adelivery agent and a TeNT substrate, where the substrate includes adonor fluorophore; an acceptor having an absorbance spectrum overlappingthe emission spectrum of the donor fluorophore; and a TeNT recognitionsequence containing a cleavage site that intervenes between the donorfluorophore and the acceptor and where, under the appropriateconditions, resonance energy transfer is exhibited between the donorfluorophore and the acceptor. A variety of TeNT substrates are useful inthe substrate invention including those having at least six consecutiveresidues of VAMP, the six consecutive residues containing Gln-Phe, or apeptidomimetic thereof. Such a TeNT substrate composition of theinvention can be, for example, a peptide or peptidomimetic having atmost twenty, thirty, forty, fifty or 100 residues. Furthermore, avariety of fluorophore-acceptor combinations are useful in a TeNTsubstrate composition of the invention; these combinations include butare not limited to fluorescein-tetramethylrhodamine, DABCYL-EDANS andAlexa Fluor®-488-QSY-7®.

In the substrate compositions of the invention, a delivery agentfacilitates uptake of a clostridial toxin substrate into a cell such asneuron or a glandular cell such as a pancreatic acinar cell. Thedelivery agent can be covalently linked to the clostridial toxinsubstrate or can be non-covalently associated with the clostridial toxinsubstrate. As non-limiting examples, delivery agents useful in thesubstrate compositions of the invention include antennapedia proteins,HIV TAT proteins, herpes simplex virus VP22 proteins, and activefragments thereof, as well as delivery agents such as Chariot™ and otherMPG peptides, each of which is discussed further hereinbelow.

As used herein, the term “delivery agent” means any molecule thatenables or enhances internalization of an associated or linkedclostridial toxin substrate into a cell. Delivery agents are known inthe art and include but are not limited to protein transduction peptidesand peptidomimetics and cell-permeant peptides and peptidomimetics. Theterm delivery agent encompasses, without limitation, proteins, peptides,peptidomimetics, small molecules, nucleic acid molecules, liposomes,lipids, viruses, retroviruses and cells. It is understood that asubstrate composition containing a delivery agent useful in theinvention generally is not retained in intracellular vesicles uponinternalization but rather is eventually delivered, for example, to thecytoplasm. Thus, the term delivery agent encompasses, withoutlimitation, molecules that transport associated or linked substrate tothe cell cytoplasm or nucleus. It further is understood that the term“delivery agent” encompasses molecules that are internalized by anymechanism, including delivery agents which function viareceptor-mediated endocytosis and those which are independent ofreceptor-mediated endocytosis.

A variety of delivery agents can be covalently linked to a clostridialtoxin substrate in a composition of the invention, including, withoutlimitation, protein transduction peptides, cell permeant peptides,phosphopeptides, peptides containing D-amino acids, and other denaturedor folded, modified or unmodified, and naturally occurring or syntheticproteins, peptides and peptidomimetics. Such delivery agents include,without limitation, nuclear and secreted proteins and active fragmentsand analogs thereof. In particular embodiments, a delivery agent usefulin the invention is a peptide or peptidomimetic having a length of lessthan 50 residues, a length of less than 40 residues, a length of lessthan 30 residues, a length of less than 20 residues, or a length of lessthan 15 residues. In a further embodiment, a delivery agent useful inthe invention is a predominantly hydrophobic peptide or peptidomimetic.In another embodiment, a delivery agent useful in the invention is apredominantly basic peptide or peptidomimetic. In yet anotherembodiment, a delivery agent useful in the invention is an α-helicalpeptide or peptidomimetic such as an amphipathic α-helical peptide orpeptidomimetic. In a further embodiment, a delivery agent useful in theinvention is an amphipathic peptide or peptidomimetic such as a basicamphipathic peptide or peptidomimetic. And, in a further embodiment, adelivery agent useful in the invention is a denatured peptide orpeptidomimetic, which is linked to a denatured or folded clostridialtoxin substrate; denaturation has been shown to facilitateinternalization as described, for example, in WO 99/55899.

As non-limiting examples, delivery agents suitable for use in theinvention when covalently linked to a clostridial toxin substrateinclude ciliary neurotrophic factor (CNTF) or an active fragmentthereof; caveolin or an active fragment thereof; interleukin-1β (IL-1β)or an active fragment thereof; thioredoxin or an active fragmentthereof; Antennapedia or an active fragment thereof such as penetratin-1(SEQ ID NO: 1); fibroblast growth factor-1 (FGF-1) or an active fragmentthereof; Engrailed or an active fragment thereof; Hoxa-5 or an activefragment thereof; Kaposi fibroblast growth factor (kFGF) or an activefragment thereof, for example, AAVALLPAVLLALLAP (SEQ ID NO: 22); humanβ3 integrin or an active fragment thereof such as a hydrophobic signalsequence; a nuclear localization sequence (NLS) such as TPPKKKRKVEDP(SEQ ID NO: 23); FGF-2 or an active fragment thereof; transportan or anactive fragment thereof such as GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:24); lactoferrin or an active fragment thereof; VP22 or an activefragment thereof; HIV type I transactivator (HIV TAT) or an activefragment thereof such as YGRKKRRQRRR (SEQ ID NO: 2); or a heat shockprotein such as HSP70 or an active fragment thereof. These andadditional delivery agents are well known in the art as described, forexample, in Ho, Cancer Res. 61:474-477 (2001); Schwarze and Dowdy,Trends Pharmacol. Sci. 21:45-48 (2000); Prochiantz, Curr. Opin. CellBiol. 12:400-406 (2000); Ford et al., Gene Therapy 8:1-4 (2001); Dunicanand Doherty, Biopolymers Peptide Sci. 60:45-60 (2001); and Schwartz andZhang, Curr. Opin. Mol. Ther. 2:162-167 (2000).

In one embodiment, the invention is practiced with a delivery agentwhich is a homeoprotein or an active fragment thereof, for example, ahomeodomain or an active fragment thereof. Homeoproteins arehelix-turn-helix proteins that contain a DNA-binding domain of about 60residues, denoted the homeodomain. A variety of homeoproteins,homeodomains and active fragments thereof can be delivery agents usefulin the invention including, without limitation, Antennapedia, Engrailed1(En1), Engrailed2 (En2), Hoxa-5, Hoxc-8, Hoxb-4 and Knotted-1 (KN1). Asan example, En1 and En1 have been expressed in COS7 cells, where theyare first secreted and then internalized by other cells. See, forexample, Prochiantz, supra, 2000.

In another embodiment, a substrate composition of the invention includesa delivery agent which is the homeodomain protein, Antennapedia, or anactive fragment thereof. Antennapedia is a member of a family ofdevelopmentally important Drosophila homeoproteins which translocateacross neuronal membranes. The third helix of the Antennapediahomeodomain, the 16 residue peptide “penetratin-1” (SEQ ID NO: 1), isinternalized into live cells. The internalization occurs both at 37° C.and 4° C., indicating that delivery is neither receptor-mediated norenergy-dependent. Additional delivery agents include peptides andpeptidomimetics related in sequence to Penetratin-1 such as, withoutlimitation, one of the peptides shown below in Table A or anotherpenetratin-derived peptide or peptidomimetic, including a retroinverseor all D-amino acid peptide or peptidomimetic, or a related butnon-α-helical peptide or peptidomimetic (see, for example, Prochiantz,supra, 2000). In one embodiment, such a penetratin-derived peptideretains the tryptophan, phenylalanine and glutamine residues ofpenetratin-1 (SEQ ID NO: 1). TABLE A Penetratin-Derived Peptides Usefulas Delivery Agents Peptide Sequence SEQ ID NO: 43-58 RQIKIWFQNRRMKWKK 158-43 KKWKMRRNQFWIKIQR 25 43-58 RQIKIWFQNRRMKWKK 26 Pro50RQIKIWFPNRRMKWKK 27 3Pro RQPKIWFPNRRMPWKK 28 Met-Arg RQIKIWFQNMRRKWKK 297Arg RQIRIWFQNRRMRWRR 30 W/R RRWRRWWRRWWRRWRR 31

In another embodiment, a substrate composition of the invention includesa delivery agent which is a HIV trans-activator (TAT) protein or anactive fragment thereof. Such a delivery agent can include, for example,a sequence identical or similar to residues 47-57 or 47-59 of HIV TAT(Schwartz et al., Science 285:1569-1572 (1999); and Ho et al., supra,2001). As an example, fusion proteins including residues 47-57 of HIVTAT (YGRKKRRQRRR; SEQ ID NO: 2) cross the plasma membrane of, forexample, human and murine cells in vitro and in vivo (Schwartz andZhang, supra, 2000); a variety of proteins from 15 to 120 KDa have beenshown to retain biological activity when fused to a HIV TAT deliveryagent. An HIV TAT delivery agent can be positively charged and canfunction, for example, in an energy-, receptor-, transporter- andendocytosis-independent manner to deliver a covalently linkedclostridial toxin substrate to, for example, 90-100% of target cells.

A substrate composition of the invention also can include as a deliveryagent a herpes simplex virus VP22 protein or active fragment thereof. Inone embodiment, a substrate composition of the invention includes an HSVtype 1 (HSV-1) VP22 protein or active fragment thereof. HSV VP22, anuclear transcription factor, can cross the plasma membrane throughnon-classical endocytosis and can enter cells independent of GAPjunctions and physical contacts. As a fusion with a variety of differentproteins, HSV VP22 results in uptake into cells of different typesincluding terminally differentiated cells (Ford et al., supra, 2001;Schwartz and Zhang, supra, 2000) and can function to deliver a linkedclostridial toxin substrate to, for example, 90-100% of cultured cells.

In another embodiment, a delivery agent useful in the inventioncorresponds to or is derived from a hydrophobic signal sequence. Such adelivery agent can be, for example, the Kaposi fibroblast growth factor(kFGF) or an active fragment thereof such as AAVALLPAVLLALLAP (SEQ IDNO: 22); human β3 integrin or an active fragment thereof; or anotherhydrophobic delivery agent such as one of those described in Dunican andDoherty, supra, 2001.

A delivery agent useful in the invention also can be a syntheticsequence that shares one or more characteristics of a naturallyoccurring delivery agent such as a protein transduction domain (PTD).Such delivery agents include, but are not limited to, L- and D-arginineoligomers, for example, 9-mers of L- or D-arginine and related peptoids(Wender et al., Proc. Natl. Acad. Sci., USA 97:13003-13008 (2000). Suchdelivery agents further include basic peptides and peptidomimetics;basic α-helical peptides and peptidomimetics; and peptides andpeptidomimetics with optimized arginine alignment or optimized α-helicalcharacter as compared to a naturally occurring protein transductiondomain such as residues 47-57 of HIV TAT. See, for example, Ho et al.,supra, 2001, and WO 99/29721. Additional non-limiting examples ofdelivery agents useful in the invention include SCWK_(n) (SEQ ID NO:32); (LARL)_(n) (SEQ ID NO: 33); HA2; RGD; K₁₆RGD (SEQ ID NO: 34);loligomer; AlkCWK₁₈ (SEQ ID NO: 35); DiCWK18 (SEQ ID NO: 36); DipaLytic;Plae (SEQ ID NO: 37); Kplae (SEQ ID NO: 38) and other delivery agentsknown in the art or which can be prepared by routine methods (see, forexample, Schwartz and Zhang, supra, 2000). The skilled personunderstands that these and other naturally occurring and syntheticdelivery agents can be useful in the substrate compositions of theinvention.

A delivery agent useful in the invention also can be an agent thatenables or enhances cellular uptake of a non-covalently associatedclostridial toxin substrate. In one embodiment, such a delivery agent ispeptide containing two independent domains: a hydrophobic domain and ahydrophilic domain. In another embodiment, such a delivery agent is anMPG peptide, which is a peptide derived from both the nuclearlocalization sequence (NLS) of SV40 large T antigen and the fusionpeptide domain of HIV-1 gp41. In a further embodiment, such a deliveryagent is an MPG peptide having the amino acid sequenceGALFLGFLGAAGSTMGAWSQPKSKRKV (SEQ ID NO: 39). In yet a furtherembodiment, such a delivery agent is an amphipathic peptide such asPep-1. These and related delivery agents that function in the absence ofcovalent linkage, sometimes known as “protein transfection products,”are well known in the art as described, for example, in Morris et al.,Nucl. Acids Res. 27:3510-3517 (1999); Morris et al., J. Biol. Chem.274:24941-24946 (1999); and Morris et al., Nature Biotech. 19:1173-1176(2001). Such peptide delivery agents can be prepared by routine methodsand are commercially available; as an example, the Chariot™ product isavailable from Active Motif (Carlsbad, Calif.).

A clostridial toxin substrate useful in the invention includes, in part,a donor fluorophore. As used herein, the term “donor fluorophore” meansa molecule that, when irradiated with light of a certain wavelength,emits light, also denoted fluorescence, of a different wavelength. Theterm fluorophore is synonymous in the art with the term “fluorochrome.”

A clostridial toxin substrate useful in the invention also includes, inpart, an acceptor. As used herein, the term “acceptor” means a moleculethat can absorb energy from, and upon excitation of, a donor fluorophoreand is a term that encompasses fluorophores as well as non-fluorescentmolecules. An acceptor useful in a clostridial toxin substrate has anabsorbance spectrum which overlaps the emission spectrum of a donorfluorophore included in the substrate. An acceptor useful in theinvention generally has rather low absorption at a wavelength suitablefor excitation of the donor fluorophore.

As set forth above, an acceptor has an absorbance spectrum that overlapsthe emission spectrum of the donor fluorophore. The term “overlapping,”as used herein in reference to the absorbance spectrum of an acceptorand the emission spectrum of a donor fluorophore, means an absorbancespectrum and emission spectrum that are partly or entirely shared. Thus,in such overlapping spectra, the high end of the range of the donorfluorophore's emission spectrum is higher than the low end of the rangeof the acceptor's absorbance spectrum.

A clostridial toxin substrate useful in the invention contains acleavage site that “intervenes” between a donor fluorophore and anacceptor having an absorbance spectrum which overlaps the emissionspectrum of the donor fluorophore. Thus, the cleavage site is positionedin between the fluorophore and acceptor such that cleavage at the siteresults in a first molecule containing the fluorophore and a secondmolecule containing the acceptor. It is understood that all or only aportion of the clostridial toxin recognition sequence can intervenebetween the donor fluorophore and acceptor.

A clostridial toxin substrate useful in the invention contains, in part,a clostridial toxin recognition sequence which includes a cleavage site.Such a clostridial toxin substrate is susceptible to cleavage by atleast one clostridial toxin under conditions suitable for clostridialtoxin protease activity.

As used herein, the term “clostridial toxin recognition sequence” meansa scissile bond together with adjacent or non-adjacent recognitionelements sufficient for detectable proteolysis at the scissile bond by aclostridial toxin under conditions suitable for clostridial toxinprotease activity. A variety of useful clostridial toxin recognitionsequences are discussed hereinbelow.

In particular embodiments, a substrate composition of the invention is achimeric protein, peptide or peptidomimetic. A substrate composition ofthe invention can be, for example, a chimeric peptide or peptidomimetichaving at most 20 residues, at most 40 residues, at most 50 residues, atmost 100 residues, at most 150 residues, at most 200 residues, at most250 residues, at most 300 residues, at most 350 residues or at most 400residues.

As used herein, the term “peptidomimetic” is used broadly to mean apeptide-like molecule that is cleaved by the same clostridial toxin asthe peptide substrate upon which it is structurally based. Suchpeptidomimetics include chemically modified peptides, peptide-likemolecules containing non-naturally occurring amino acids, and peptoids,which are peptide-like molecules resulting from oligomeric assembly ofN-substituted glycines, and are cleaved by the same clostridial toxin asthe peptide substrate upon which the peptidomimetic is derived (see, forexample, Goodman and Ro, Peptidomimetics for Drug Design, in “Burger'sMedicinal Chemistry and Drug Discovery” Vol. 1 (ed. M. E. Wolff; JohnWiley & Sons 1995), pages 803-861).

A variety of peptidomimetics are known in the art including, forexample, peptide-like molecules which contain a constrained amino acid,a non-peptide component that mimics peptide secondary structure, or anamide bond isostere. A peptidomimetic that contains a constrained,non-naturally occurring amino acid can include, for example, anα-methylated amino acid; an α,α-dialkyl-glycine or α-aminocycloalkanecarboxylic acid; an N^(α)—C^(α) cylized amino acid; an N^(α)-methylatedamino acid; a β- or γ-amino cycloalkane carboxylic acid; anα,β-unsaturated amino acid; a β,β-dimethyl or β-methyl amino acid; aβ-substituted-2,3-methano amino acid; an NC^(δ) or C^(α)—C^(δ) cyclizedamino acid; or a substituted proline or another amino acid mimetic. Inaddition, a peptidomimetic which mimics peptide secondary structure cancontain, for example, a nonpeptidic β-turn mimic; γ-turn mimic; mimic ofβ-sheet structure; or mimic of helical structure, each of which is wellknown in the art. A peptidomimetic also can be a peptide-like moleculewhich contains, for example, an amide bond isostere such as aretro-inverso modification; reduced amide bond; methylenethioether ormethylenesulfoxide bond; methylene ether bond; ethylene bond; thioamidebond; trans-olefin or fluoroolefin bond; 1,5-disubstituted tetrazolering; ketomethylene or fluoroketomethylene bond or another amideisostere. One skilled in the art understands that these and otherpeptidomimetics are encompassed within the meaning of the term“peptidomimetic” as used herein.

Further provided herein is a cell containing a clostridial toxinsubstrate that includes a donor fluorophore; an acceptor having anabsorbance spectrum overlapping the emission spectrum of the donorfluorophore; and a clostridial toxin recognition sequence containing acleavage site that intervenes between the donor fluorophore and theacceptor, where resonance energy transfer is exhibited between the donorfluorophore and the acceptor under the appropriate conditions. In oneembodiment, the cell is a transfected cell. In another embodiment, thecell is a stably transfected cell. A variety of cells are useful in theinvention including, without limitation, primary cells; establishedcells; human cells; neuronal cells such as primary neurons, establishedneurons and human neurons; and non-neuronal cells, which can be, forexample, glandular cells such as pancreatic acinar cells. Neurons usefulin the invention include CNS neurons and peripheral neurons; asnon-limiting examples, such neurons include neuroblastoma cells, spinalcord neurons, dorsal root ganglion neurons, cerebral cortex neurons,cerebellar neurons, hippocampal neurons and motor neurons.

In a cell of the invention, the clostridial toxin substrate optionallycan be covalently linked to a delivery agent. Such a delivery agent canbe, for example, a protein, peptide or peptidomimetic. A variety ofdelivery agents can be useful in a cell of the invention including,without limitation, an antennapedia protein or active fragment thereof,such as an active fragment having the amino acid sequenceRQIKIWFQNRRMKWKK (SEQ ID NO: 1); an HIV TAT protein or active fragmentthereof, such as an active fragment having the amino acid sequenceYGRKKRRQRRR (SEQ ID NO: 2); or a herpes simplex virus VP22 protein oractive fragment thereof, such as a herpes simplex virus VP22 proteinhaving the amino acid sequence SEQ ID NO: 3, or active fragment thereof.In one embodiment, the invention provides a cell containing a chimericprotein, peptide or peptidomimetic that includes a clostridial toxinsubstrate and a delivery agent operatively fused to the substrate. Sucha chimeric protein, peptide or peptidomimetic can have, for example, alength of at most 50 or 100 residues.

A variety of clostridial toxin substrates can be included in a cell ofthe invention. Such clostridial toxin substrates include botulinum toxinsubstrates of all serotypes as well as tetanus toxin substrates. In oneembodiment, the invention provides a cell containing a BoNT/A substratethat includes, in part, a BoNT/A recognition sequence. Such a BoNT/Asubstrate can include, for example, at least six consecutive residues ofSNAP-25, the six consecutive residues containing Gln-Arg, or apeptidomimetic thereof. In another embodiment, the invention provides acell containing a BoNT/B substrate which includes, in part, a BoNT/Brecognition sequence. BoNT/B substrates useful in the cells of theinvention include, without limitation, those having at least sixconsecutive residues of VAMP, the six consecutive residues containingGln-Phe, or a peptidomimetic thereof. In a further embodiment, theinvention provides a cell containing a BoNT/C1 substrate that includes,in part, a BoNT/C1 recognition sequence; BoNT/C1 substrates useful inthe cells of the invention encompass those having at least sixconsecutive residues of syntaxin, the six consecutive residuescontaining Lys-Ala, or a peptidomimetic thereof, and those including atleast six consecutive residues of SNAP-25, the six consecutive residuescontaining Arg-Ala, or a peptidomimetic thereof. In another embodiment,the invention provides a cell containing a BoNT/D substrate thatincludes, in part, a BoNT/D recognition sequence. A variety of BoNT/Dsubstrates are useful in the cells of the invention including, yet notlimited to, BoNT/D substrates having at least six consecutive residuesof VAMP, the six consecutive residues containing Lys-Leu, or apeptidomimetic thereof.

In a further embodiment, the invention provides a cell containing aBoNT/E substrate that includes, in part, a BoNT/E recognition sequence.Such a BoNT/E substrate can have, for example, at least six consecutiveresidues of SNAP-25, the six consecutive residues containing Arg-Ile, ora peptidomimetic thereof. In an additional embodiment, there is providedherein a cell containing a BoNT/F substrate that includes, in part, aBoNT/F recognition sequence. BoNT/F substrates useful in the cells ofthe invention can have, for example, at least six consecutive residuesof VAMP, the six consecutive residues containing Gln-Lys, or apeptidomimetic thereof. In still a further embodiment, the inventionprovides a cell containing a BoNT/G substrate that includes, in part, aBoNT/G recognition sequence. Useful BoNT/G substrates include, yet arenot limited to, those having at least six consecutive residues of VAMP,the six consecutive residues containing Ala-Ala, or a peptidomimeticthereof. In a further embodiment, the present invention provides a cellcontaining a TeNT substrate which includes, in part, a TeNT recognitionsequence. A variety of TeNT substrates are useful in the inventionincluding those having at least six consecutive residues of VAMP, thesix consecutive residues containing Gln-Phe, or a peptidomimeticthereof.

As described further herein, a variety of donor fluorophores andacceptors are useful in the cells of the invention. Donor fluorophoresuseful in the invention include, without limitation, Alexa Fluor®-488,DABCYL and BODIPY®. Acceptors useful in the invention includenon-fluorescent acceptors as well as acceptor fluorophores, includingacceptor fluorophores having a fluorescence lifetime of at least 1microsecond. Acceptors useful in the cells of the invention furtherinclude tetramethylrhodamine, EDANS and QSY-7®.

The term “cell,” as used herein, means any eukaryotic cell thatexpresses, or can be engineered to express, at least one receptor thatbinds a clostridial toxin. The term cell encompasses, withoutlimitation, primary cells; cultured cells; established cells; normalcells; transformed cells; tumor cells; infected cells; stably ortransiently transfected cells, including stably and transientlytransfected cells; and proliferating and terminally differentiatedcells, as well as cells of a variety of species and cell types. Thus,the term cell encompasses, without limitation, mammalian cells such asmurine, rat, porcine, bovine, equine, primate and human cells. It isunderstood that cells useful in the invention can be in any state suchas proliferating or quiescent; intact or permeabilized such as throughelectroporation or treatment with digitonin; and further can be inisolated form or part of a heterogeneous cell population, tissue ororganism. It further is understood that cells useful in the inventioninclude those which express a clostridial toxin substrate under controlof a constitutive or inducible promoter and that these and other cellsuseful in the invention can express one or more endogenous clostridialtoxin target proteins or can express low or undetectable levels of oneor all target proteins such as SNAP-25, VAMP and syntaxin.

Cells useful in the invention include those that express one or moreendogenous low or high affinity clostridial toxin receptors; cells thatexpress low or undetectable levels of endogenous receptor and that havebeen transfected with, or otherwise engineered to express, one or moreexogenous nucleic acid molecules encoding one or more clostridial toxinreceptors; and cells that express a combination of endogenous andexogenous toxin receptors for one or more clostridial toxin serotypes.It is understood that the selection of a cell depends, in part, on whichclostridial toxin is to be assayed. As an example, to assay for BoNT/Aactivity, one selects a cell that expresses or can be engineered toexpress a low or high affinity receptor for BoNT/A.

In one embodiment, the invention provides a neuron containing aclostridial toxin substrate that includes a donor fluorophore, anacceptor having an absorbance spectrum overlapping the emission spectrumof the donor fluorophore, and a clostridial toxin recognition sequenceincluding a cleavage site, where the cleavage site intervenes betweenthe donor fluorophore and the acceptor and where resonance energytransfer is exhibited between the donor fluorophore and the acceptorunder the appropriate conditions.

A variety of neurons can be useful in the invention. As non-limitingexamples, a neuron useful in the invention can be a primary neuron;established neuron; transformed neuron; stably transfected neuron; ormotor or sensory neuron, and further can be, for example, a mammalian,murine, rat, primate or human neuron. A neuron useful in the inventioncan be a peripheral neuron or CNS neuron; as non-limiting examples,spinal cord neurons such as an embryonic spinal cord neurons, dorsalroot ganglia (DRG) neurons, cerebral cortex neurons, cerebellar neurons,hippocampal neurons and motor neurons can be useful in the invention asdescribed further below.

Exemplary neurons useful in the invention include, but are not limitedto, primary cultures of embryonic DRG neurons, for example, primarycultures of embryonic rat DRG neurons as described in Welch et al.,Toxicon 38:245-258 (2000); and primary cultures of fetal spinal cordneurons, for example, primary cultures of murine fetal spinal cordneurons as described in Neale et al., J. Cell Biol. 147:1249-1260(1999), or Chaddock et al., Infect. Immun. 68:2587-2593 (2000)).

Exemplary neuronal cell lines useful in the invention include, withoutlimitation, neuroblastoma cell lines such as LA-N-2, SH—SY5Y, N2a,NS-20Y and NIE-115; hybrid cell lines, including neuroblastoma/gliomahybrids such as NG108-C15; motor neuron cell lines such as NSC-34 andNSC-19; spinal cord cell lines such as M4b; CNS cell lines; cerebralcortex cell lines such as CNh; dorsal root ganglion cell lines such asG4b; hippocampal cell lines such as HT-22; and pheochromocytoma celllines such as PC12.

A neuronal cell line useful in the invention can be, for example, aneuroblastoma cell line such as a murine, primate or human neuroblastomacell line. Exemplary neuroblastoma cell lines useful in the inventioninclude, without limitation, LA-N-2, SH—SY5Y, N2a, NS-20Y and NIE-115.As an example, the invention can be practiced with the LA-N-2 humanneuroblastoma cell line, which has properties of cholinergic neurons andexpresses well characterized cholinergic markers (Rylett et al., J.Neurochem. 61:1388-1397 (1993); Singh et al., J. Neurosci. Res.25:476-485 (1990); and Yeh et al., Neuroscience 27:309-315 (1988)). As afurther example, the invention can be practiced with the SH—SY5Y humanneuroblastoma cell line, which exhibits inhibition of [³H]-noradrenalinerelease induced by K⁺/Ca²⁺ upon exposure to botulinum toxin (Purkiss etal., Neurotoxicology 22:447-453 (2001)).

Hybrid neuronal cell lines such as murine, primate and human hybridneuronal cell lines also can be useful in the invention. Such hybridcell lines include neuroblastoma hybrids such as neuroblastoma/gliomahybrids. As an example, the NG108-C15 cell line is a hybrid of mouseneuroblastoma and rat glioma cells that can be useful in the invention(Yokosawa et al., Infect. Immun. 57:272-277 (1989); Yokosawa et al.,Toxicon 29:261-264 (1991)). The NG108-C15 cell line can be engineered toinclude, for example, a BoNT/C1 substrate to assay for BoNT/C1 activity.Additional hybrid cell lines include NSC cell lines, which are hybridsof neuroblastomas and spinal cord neurons that resemble developing motorneurons (Cashman et al., Dev. Dyn. 194:209-221 (1992)).

A neuronal cell line useful in the invention also can be a motor neuroncell line such as a murine, primate or human motor neuron cell line.NSC-34 and NSC-19 are exemplary motor neuron cell lines useful in theinvention; these cell lines, which are clonal hybrids of mouseneuroblastoma (N18TG2) and isolated embryonic (day 12-14) mouse spinalcord motor neurons, express motor neuron characteristics and display amultipolar neuron-like phenotype (Eggett et al., J. Neurochem.74:1895-1902 (2000)). NSC-34 and NSC-19 cells express high levels ofcholine acetyltransferase (CHAT), a marker of motor neurons. These cellsalso generate action potentials; express neurofilament triplet proteins;and synthesize, store and release acetylcholine.

A neuronal cell line useful in the invention also can be a spinal cordcell line such as a murine, primate or human spinal cord cell line. Asan example, a human spinal cord cell line can be generated fromprecursors of human embryonic spinal cord cells (first trimesterembryos) that are immortalized with a tetracycline repressible v-myconcogene as described in Li et al., J. Neurosci. Res. 59:342-352 (2000).Such cells can be expanded indefinitely in proliferative growthconditions before rapid differentiation (4-7 days) into functionalneurons that express neuronal phenotypic markers such as cholineacetyltransferase. As another example, a murine spinal cord cell linecan be prepared by immortalizing an embryonic spinal cord culture usingtransforming media. Such a spinal cord cell line can be, for example,the murine M4b line and can express neuronal markers such as NSE,synaptophysin, MAP-2, and choline acetyltransferase and releaseacetylcholine upon appropriate stimulation (Cardenas et al., J.Neurosci. Res. 68:46-58 (2002)).

Human central nervous system (CNS) cell lines, including murine, primateand human CNS cell lines, also can be useful in the invention. A usefulCNS cell line can be, for example, a human CNS cell line immortalizedwith a tetracycline repressible v-myc oncogene as described in Sah etal., Nature Biotechnol. 15:574-580 (1997). Upon repression of theoncogene, the cells differentiate into neurons.

Cerebral cortex (CNh) cell lines also are neurons useful in theinvention. Useful cerebral cortex cell lines include, but are notlimited to, murine, primate and human cell lines. As an example, murinecortex primary cultures from 12-16 days embryos can be immortalized, forexample, by culturing the cells in conditioned media from a rat thyroidcell line that induces transformation in vitro. The immortalized cellscan be differentiated into neurons expressing neuronal markers using theappropriate media; these differentiated cells express cholineacetyltransferase and secrete acetylcholine and glutamate in response todepolarization and nicotine stimulation (Allen et al., Eur. J. Neurosci.12:3259-3264 (2000)).

Dorsal root ganglia cell lines including murine, primate and humandorsal root ganglia cell lines also can be useful in the invention.Embryonic dorsal root ganglia primary cultures can be immortalized withtransforming conditioned media as described above. Upon differentiation,the cell line exhibits neuronal traits and lacks glial markers byimmunohistochemistry. Release of neurotransmitters such as acetylcholinecan be induced in response to potassium and nicotine (Allen et al.,Neuroreport 13:491-496 (2002)). An examplary DRG cell line useful in theinvention is the murine DRG cell line G4b.

The invention also can be practiced with hippocampal cell lines,including murine, primate and human hippocampal lines. As a non-limitingexample, the murine hippocampal cell line HT-22 can be useful in theinvention. The skilled person understands that these and additionalprimary and established neurons can be useful in the compositions andmethods of the invention.

It is understood that the invention can be practiced with both intactand permeabilized cells. In one embodiment, a cell of the invention ispermeabilized, for example, through electroporation or exposure todigitonin or low ionic strength buffer. In one further embodiment, acell of the invention is a permeabilized PC12 cell.

As discussed above, it is understood that a neuron useful in theinvention expresses endogenous or exogenous low or high affinityreceptors for one or more clostridial toxins. Such a neuron alsogenerally exhibits inhibition of exocytosis upon exposure to clostridialtoxin with, for example, an IC₅₀ of less than 50 nM, less than 5 nM,less than 0.5 nM, less than 0.05 nM, less than 0.005 nM, less than0.0005 nM, less than 0.00005 nM or less than 0.000005 nM. In particularembodiments, the invention provides a neuron containing a BoNT/Asubstrate which exhibits inhibition of exocytosis with an IC₅₀ of lessthan 50 nM, less than 5 nM, less than 0.5 nM, less than 0.05 nM, lessthan 0.005 nM, less than 0.0005 nM, less than 0.00005 nM or less than0.000005 nM upon exposure to BoNT/A. In further embodiments, theinvention provides a neuron containing a BoNT/B substrate which exhibitsinhibition of exocytosis with an IC₅₀ of less than 50 nM, less than 5nM, less than 0.5 nM, less than 0.05 nM, less than 0.005 nM, less than0.0005 nM, less than 0.00005 nM or less than 0.000005 nM upon exposureto BoNT/B. In other embodiments, the invention provides a neuroncontaining a BoNT/C1 substrate which exhibits inhibition of exocytosiswith an IC₅₀ of less than 50 nM, less than 5 nM, less than 0.5 nM, lessthan 0.05 nM, less than 0.005 nM, less than 0.0005 nM, less than 0.00005nM or less than 0.000005 nM upon exposure to BoNT/C1. In still furtherembodiments, the invention provides a neuron containing a BoNT/Dsubstrate which exhibits inhibition of exocytosis with an IC₅₀ of lessthan 50 nM, less than 5 nM, less than 0.5 nM, less than 0.05 nM, lessthan 0.005 nM, less than 0.0005 nM, less than 0.00005 nM or less than0.000005 nM upon exposure to BoNT/D. In additional embodiments, theinvention provides a neuron containing a BoNT/E substrate which exhibitsinhibition of exocytosis with an IC₅₀ of less than 50 nM, less than 5nM, less than 0.5 nM, less than 0.05 nM, less than 0.005 nM, less than0.0005 nM, less than 0.00005 nM or less than 0.000005 nM upon exposureto BoNT/E. In yet further embodiments, the invention provides a neuroncontaining a BoNT/F substrate which exhibits inhibition of exocytosiswith an IC₅₀ of less than 50 nM, less than 5 nM, less than 0.5 nM, lessthan 0.05 nM, less than 0.005 nM, less than 0.0005 nM, less than 0.00005nM or less than 0.000005 nM upon exposure to BoNT/F. In furtherembodiments, the invention provides a neuron containing a BoNT/Gsubstrate which exhibits inhibition of exocytosis with an IC₅₀ of lessthan 50 nM, less than 5 nM, less than 0.5 nM, less than 0.05 nM, lessthan 0.005 nM, less than 0.0005 nM, less than 0.00005 nM or less than0.000005 nM upon exposure to BoNT/G. In still further embodiments, theinvention provides a neuron containing a TeNT substrate which exhibitsinhibition of exocytosis with an IC₅₀ of less than 50 nM, less than 5nM, less than 0.5 nM, less than 0.05 nM, less than 0.005 nM, less than0.0005 nM, less than 0.00005 nM or less than 0.000005 nM upon exposureto TeNT. It is understood that the same neuron can express two or morereceptors for different clostridial toxin serotypes, with the same or adifferent IC₅₀ for each individual toxin serotype.

A variety of non-neuronal cells including primary and established cellsalso are useful in the invention. Such non-neuronal cells encompassprimary cells; established cells; stably and transiently transfectedcells; and tumor cells as well as cells of all species of originincluding mammalian, murine, rat, primate and human cells. Asnon-limiting examples, non-neuronal cells useful in the inventioninclude glandular cells such as pancreatic acinar cells, pancreaticβ-islet cells, and insulinoma HIT or INS-1 cells; fibroblasts; musclecells; and hepatocytes.

Non-neuronal cells useful in the invention further include, withoutlimitation, any of the following primary or established cells: anteriorpituitary cells; adrenal cells such as chromaffin cells of the adrenalmedulla; stomach cells such as enterochromaffin-like cells; pancreaticcells such as pancreatic islet β-cells; ovarian cells such assteroid-producing ovarian cells; kidney cells such as inner medullarycollecting duct (IMCD) cells; pancreatic acinar cells; platelets;neutrophils; eosinophils; mast cells; epithelial cells such as those ofthe apical plasma membrane; and cells involved in glucose transporter(GLUT4) translocation. As non-limiting examples, a non-neuronal celluseful in the invention can include a clostridial toxin substrate whichhas a SNAP-25 recognition sequence; such a non-neuronal cell can be, forexample, a primary or established anterior pituitary cell; adrenal cellsuch as a chromaffin cell of the adrenal medulla; stomach cell such asan enterochromaffin-like cell; pancreatic cell such as a pancreaticislet β-cell; or ovarian cell such as a steroid-producing ovarian cell.As further non-limiting examples, a non-neuronal cell useful in theinvention can include a clostridial toxin substrate which has a SNAP-23recognition sequence; such a non-neuronal cell can be, for example, akidney cell such as an inner medullary collecting duct (IMCD) cell; apancreatic acinar cell; a platelet; a neutrophil; an eosinophil; a mastcell; an epithelial cell such as one of the apical plasma membrane; or acell involved in glucose transporter (GLUT4) translocation. It isunderstood that these and a variety of other primary and establishednon-neuronal cells can be useful in the invention.

In one embodiment, the invention provides an established non-neuronalcell that includes a nucleic acid molecule encoding a clostridial toxinsubstrate that contains a donor fluorophore, an acceptor having anabsorbance spectrum overlapping the emission spectrum of the donorfluorophore and a clostridial toxin recognition sequence containing acleavage site, where the cleavage site intervenes between the donorfluorophore and the acceptor and where, under the appropriateconditions, resonance energy transfer is exhibited between the donorfluorophore and the acceptor. Such an established non-neuronal cell canbe, for example, stably transfected with a nucleic acid moleculeencoding a clostridial toxin substrate.

It is understood that cells that express endogenous or transfectedclostridial toxin receptor can be identified by routine methodsincluding direct and indirect assays for toxin uptake. Such methods canrely, for example, on labelled toxin such as fluorescently labeled orradiolabeled toxin. Such methods also can be performed, for example,with anti-toxin antibodies, which can be used to detect intracellulartoxin, for example, by immunocytochemistry or western blotting. Inaddition, cells that express clostridial toxin receptor and, therefore,take up one or more clostridial toxins also can be identified byassaying for cleaved target protein. As an example, a western blot usingan antibody that specifically recognizes SNAP-25₁₉₇, the cleaved productof BoNT/A, can be used to assay for uptake of BoNT/A. Further well knownassays include detection of ³H-noradrenaline or other neurotransmittersecretion as a measure of inhibition of exocytosis in neurons, or therelease of hormones from endocrine cells such as anterior pituitarycells or ovarian cells. It is understood that these and similar assaysfor intracellular toxin, toxin cleavage product, or toxin function canbe useful in selecting a neuron or other cell useful in the compositionsand methods of the invention.

The invention further provides a cell that incorporates a “composite”clostridial toxin substrate. Such a composite clostridial toxinsubstrate contains (a) a first member of a donor fluorophore-acceptorpair linked to a first partner of an affinity couple; and (b) aclostridial toxin recognition sequence containing a cleavage site, wherethe recognition sequence is linked to a second member of the donorfluorophore-acceptor pair and a second partner of the affinity couple,where the cleavage site intervenes between the second member of thedonor fluorophore-acceptor pair and the second partner of the affinitycouple, and where (a) and (b) are stably associated such that, under theappropriate conditions, resonance energy transfer is exhibited betweenthe first and second members of the donor fluorophore-acceptor pair.Thus, a composite clostridial toxin substrate is, in effect, a bipartiteclostridial toxin substrate in which the two parts are stably associatedthrough the affinity couple. As for other clostridial toxin substrates,resonance energy transfer is altered upon cleavage of the compositesubstrate. It is understood that the clostridial toxin recognitionsequences and cleavage sites described herein and well known in the artcan be useful in composite clostridial toxin substrates.

The term “donor fluorophore-acceptor pair,” as used herein, means adonor fluorophore and an acceptor that has an absorbance spectrumoverlapping the emission spectrum of the donor fluorophore. Where thefirst member of the pair is a donor fluorophore, the second member ofthe pair will be an acceptor. Where the first member of the pair is anacceptor, the second member of the pair will be a donor fluorophore.

In one embodiment, the first member of the donor fluorophore-acceptorpair is a donor fluorophore, and the second member is an acceptor. Inanother embodiment, the first member of the donor fluorophore-acceptorpair is an acceptor, and the second member is a donor fluorophore. Avariety of donor fluorophores and acceptors can be incorporated into acomposite clostridial toxin substrate useful in a cell or method of theinvention, including the donor fluorophores and acceptors describedhereinbelow. In one embodiment, the donor fluorophore is a fluorescentprotein. In another embodiment, the donor fluorophore and acceptor eachis a fluorescent protein. Useful fluorescent proteins include but arenot limited to green fluorescence protein (GFP), blue fluorescenceprotein (BFP), cyan fluorescence protein (CFP), yellow fluorescenceprotein (YFP) and red fluorescence protein (RFP).

The term “affinity couple,” as used herein, means two molecules that arecapable of forming a stable, non-covalent association. Affinity couplesuseful in a composite substrate include, without limitation,SNAP-25-syntaxin; VAMP-synaptotagmin; streptavidin-biotin; S peptide-Sprotein; receptor-ligand; dimeric receptors or other interactingproteins. In one embodiment, the affinity couple is SNAP-25-syntaxin. Inanother embodiment, the affinity couple is VAMP-synaptotagmin.

The present invention also provides a nucleic acid molecule that encodesa clostridial toxin substrate including a donor fluorophore; an acceptorhaving an absorbance spectrum overlapping the emission spectrum of thedonor fluorophore; and a clostridial toxin recognition sequencecontaining a cleavage site that intervenes between the donor fluorophoreand the acceptor, where resonance energy transfer is exhibited betweenthe donor fluorophore and the acceptor under the appropriate conditions.The nucleic acid molecule encoding a clostridial toxin substrate can belinked, for example, to a regulatory element such as a constitutiveregulatory element or inducible regulatory element such as, withoutlimitation, a tetracycline regulated regulatory element or ecdysoneinducible regulatory element. Genetically encoded donor fluorophores andacceptors useful in a genetically encoded clostridial toxin substrate ofthe invention include green fluorescence protein (GFP) and otherfluorescent proteins disclosed herein below and known in the art.

In one embodiment, the encoded clostridial toxin recognition sequence isresidues 34 to 206 of human SNAP-25 (SEQ ID NO: 4). In anotherembodiment, the encoded clostridial toxin recognition sequence isresidues 34 to 206 of human SNAP-25 (SEQ ID NO: 4) and the donorfluorophore or acceptor is a green fluorescence protein. In otherembodiments, the encoded clostridial toxin recognition sequence isresidues 34 to 206 of human SNAP-25 (SEQ ID NO: 4), and the donorfluorophore and acceptor each are a green fluorescence protein, bluefluorescence protein, cyan fluorescence protein, yellow fluorescenceprotein, or red fluorescence protein.

A nucleic acid molecule of the invention can further optionally includeany of a variety of constitutive or inducible regulatory elements suchas promoters or enhancers. Inducible expression systems have theadvantage that they can produce controlled intracellular levels ofsubstrate. Constitutive regulatory elements useful in the inventioninclude, without limitation, the cytomegalovirus (CMV), herpes simplexvirus thymidine kinase (HSV TK), simian virus 40 (SV40) early, 5′ longterminal repeat (LTR), elongation factor-1α (EF-1α) and polybiquitin(UbC) regulatory elements. Inducible regulatory elements useful in theinvention include tetracycline inducible and tetracycline repressibleelements such as Tet-On™ and Tet-Off™ (BD Biosciences);ecdysone-inducible elements and GAL4 regulated elements such as theGeneSwitch™ system (Invitrogen). The skilled person understands thatthese and other constitutive and inducible regulatory elements can beincluded a nucleic acid molecule of the invention.

Any of a variety of genetically encoded donor fluorophores and acceptorsare useful in the nucleic acid molecules and cells of the invention.Such donor fluorophores and acceptors include genetically encoded dyessuch as a green fluorescence protein (GFP), blue fluorescence protein(BFP), cyan fluorescence protein (CFP), yellow fluorescence protein(YFP) or red fluorescence protein such as dsRed (BD BiosciencesClontech; Palo Alto, Calif.). Such genetically encoded donorfluorophores and acceptors are well known in the art as described, forexample, in Selvin, supra, 2000, and Mahajan et al., Chemistry andBiology 6:401-409 (1999). As an example, CFP has an excitation maxima at433 nm and an emission maxima at 476 nm and can be used as a donorfluorophore in combination with YFP as an acceptor (emission maxima at527 nm). If desired, BFP can be used as a donor fluorophore incombination with GFP as the acceptor, or CFP can be used as the donorfluorophore in combination with YFP as the acceptor. Additionalgenetically encoded donor fluorophores and acceptors including Aequorearelated fluorescent proteins are well known in the art, as described,for example, in U.S. Pat. No. 5,981,200.

The invention further provides a cell that includes a geneticallyencoded clostridial toxin substrate. Thus, the invention provides a cellwhich includes a nucleic acid molecule encoding a clostridial toxinsubstrate that includes a donor fluorophore; an acceptor having anabsorbance spectrum overlapping the emission spectrum of the donorfluorophore; and a clostridial toxin recognition sequence containing acleavage site that intervenes between the donor fluorophore and theacceptor, where resonance energy transfer is exhibited between the donorfluorophore and the acceptor under the appropriate conditions. Thenucleic acid molecule can encode, for example, residues 34 to 206 ofhuman SNAP-25 (SEQ ID NO: 4), and can further include, for example, adonor fluorophore such as a green fluorescence protein, bluefluorescence protein, cyan fluorescence protein, yellow fluorescenceprotein, or red fluorescence protein, as well as an acceptor such as agreen fluorescence protein, blue fluorescence protein, cyan fluorescenceprotein, yellow fluorescence protein, or red fluorescence protein.

Any of a variety of cells can be engineered to include a nucleic acidmolecule encoding a clostridial toxin substrate, including, but notlimited to, human cells, neuronal cells and non-neuronal cells. In oneembodiment, the nucleic acid molecule encoding the clostridial toxinsubstrate is stably transfected into the cell. In another embodiment,the nucleic acid molecule encoding a clostridial toxin substrate islinked to a regulatory element such as a constitutive regulatory elementor inducible regulatory element. A variety of inducible regulatoryelements are useful in the invention, including, without limitation,tetracycline regulated regulatory elements and ecdysone inducibleregulatory elements. A genetically encoded clostridial toxin substrateof the invention can include any of a variety of genetically encodeddonor fluorophores or acceptors such as GFP.

A cell containing a nucleic acid molecule encoding a clostridial toxinsubstrate can be prepared by any of a variety of routine methodsincluding well-known transient and stable transfection methods. Asnon-limiting examples, routine techniques for introducing a nucleic acidmolecule into a cell, including a neuronal or non-neuronal cell, includemicroinjection, electroporation, lipofection, calcium-phosphate mediatedtransfection, DEAE-Dextran-mediated transfection, polybrene- orpolylysine-mediated transfection, and conjugation to an antibody,gramacidin S, artificial viral envelope or other intracellular carriersuch as TAT. See Cibelli et al., Nat. Biotech. 16:642-646 (1998); Lamband Gearhart, Cur. Opin. Gen. Dev. 5:342-348 (1995); Choi (U.S. Pat. No.6,069,010); and Current Protocols in Molecular Biology, John Wiley andSons, pp 9.16.4-9.16.11 (2000).

The present invention also provides kits for determining clostridialtoxin activity in a sample. Such kits contain a substrate composition orcell of the invention in a vial or other container and generally alsoinclude instructions for use. In one embodiment, a kit of the inventionfurther includes as a positive control a known amount of the botulinumor tetanus toxin capable of cleaving the clostridial toxin substratewhich incorporated into the substrate composition or cell included inthe kit. In another embodiment, the kit contains a substrate compositionof the invention and further includes one or both cleavage products as apositive control. Such a kit can include, for example, a substratecomposition of the invention and the corresponding cleavage productcontaining the donor fluorophore as a positive control. Where theinvention provides a kit containing a cell of the invention and apositive control, the positive control is understood to be a cell of thesame cell type, having one or both corresponding cleavage products inplace of the uncleaved clostridial toxin substrate. Such a positivecontrol cell can contain, for example, the cleavage product whichincludes the donor fluorophore.

As described further below, a combination of cells of the same ordifferent types containing different clostridial toxin substrates can beuseful for detecting the activity of two or more clostridial toxins.Thus, in one embodiment, the invention provides a kit for determiningclostridial toxin activity that includes at least two substratecompositions of the invention having recognition sequences for twodifferent clostridial toxins. In another embodiment, the inventionprovides a kit for determining clostridial toxin activity that includesat least two cells of the invention containing two different clostridialtoxin substrates having recognition sequences for two differentclostridial toxins.

The present invention also provides a method of determining clostridialtoxin activity by (a) contacting with a sample a cell containing aclostridial toxin substrate that includes a donor fluorophore; anacceptor having an absorbance spectrum overlapping the emission spectrumof the donor fluorophore; and a clostridial toxin recognition sequencecontaining a cleavage site that intervenes between the donor fluorophoreand the acceptor, where resonance energy transfer is exhibited betweenthe donor fluorophore and the acceptor under the appropriate conditions;(b) exciting the donor fluorophore; and (c) determining resonance energytransfer of the contacted cell relative to a control cell, where adifference in resonance energy transfer of the contacted cell ascompared to the control cell is indicative of clostridial toxinactivity.

The methods of the invention can be advantageously practiced to assay aclostridial toxin for several different steps required for toxinactivity. Thus, a method of the invention can be used to determine if aclostridial toxin has a functional binding domain for cellular uptake; afunctional translocation domain for delivery of light chain to the cellcytosol; and a functional proteolytic domain. In the absence of any ofthese three functions, method of the invention generally yields anegative result.

In the methods of the invention, a clostridial toxin substrateoptionally can be covalently linked to a delivery agent, which can be,for example, a protein, peptide or peptidomimetic. Useful deliveryagents include, for example, antennapedia proteins or active fragmentsthereof, such as active fragments having the amino acid sequenceRQIKIWFQNRRMKWKK (SEQ ID NO: 1); HIV TAT proteins or active fragmentsthereof, such as active fragments having the amino acid sequenceYGRKKRRQRRR (SEQ ID NO: 2); and herpes simplex virus VP22 proteins oractive fragments thereof, such as herpes simplex virus VP22 proteinshaving the amino acid sequence SEQ ID NO: 3, or active fragmentsthereof. In one embodiment, a method of the invention is practiced witha cell containing a chimeric protein, peptide or peptidomimetic thatincludes a clostridial toxin substrate and a delivery agent operativelyfused to the substrate. Such a chimeric protein, peptide orpeptidomimetic can have, for example, a length of at most 50 or 100residues.

A clostridial toxin substrate useful in a method of the invention can bea botulinum toxin substrate of any serotype or a tetanus toxinsubstrate. Thus, a method of the invention can be practiced with a cellcontaining a BoNT/A substrate which includes a BoNT/A recognitionsequence; a cell containing a BoNT/B substrate that includes a BoNT/Brecognition sequence; a cell containing a BoNT/C1 substrate thatincludes a BoNT/C1 recognition sequence; a cell containing a BoNT/Dsubstrate which includes a BoNT/D recognition sequence; a cellcontaining a BoNT/E substrate that includes a BoNT/E recognitionsequence; a cell containing a BoNT/F substrate which includes a BoNT/Frecognition sequence; a cell containing a BoNT/G substrate whichincludes a BoNT/G recognition sequence; or a cell containing a TeNTtoxin substrate that includes a TeNT recognition sequence. It isunderstood that a variety of donor fluorophores and acceptors can beincorporated into a clostridial toxin substrate useful in a method ofthe invention. As non-limiting examples, useful donor fluorophoresinclude Alexa Fluor®-488, DABCYL and BODIPY®, and useful acceptorsinclude EDANS, QSY-7® and tetramethylrhodamine. As described furtherherein, acceptors useful in the methods of the invention encompassnon-fluorescent acceptors as well as acceptor fluorophores, includingacceptor fluorophores having a relatively long fluorescence lifetime ofat least 1 microsecond.

A variety of cells are useful in the methods of the invention including,without limitation, primary cells; established cells; human cells;neurons such as primary neurons, established neurons and human neurons;and non-neuronal cells such as pancreatic acinar cells. Neurons usefulfor determining clostridial toxin activity according to a method of theinvention include central nervous system neurons and peripheral neurons;as non-limiting examples, such a neuron can be a neuroblastoma cell,spinal cord neuron, dorsal root ganglion neuron, cerebral cortex neuron,cerebellar neuron, hippocampal neuron or motor neuron.

One skilled in the art understands that a variety of samples can beassayed for clostridial toxin activity according to a method of theinvention. Such samples include, without limitation, crude cell lysates;isolated clostridial toxins; formulated clostridial toxin products suchas BOTOX-; and foodstuffs.

A variety of means can be used to determine resonance energy transfersubsequent to excitation of a donor fluorophore in a method of theinvention. In one embodiment, a method of the invention includes thestep of detecting donor fluorescence intensity of the contacted cellrelative to a control cell, where increased donor fluorescence intensityof the contacted cell as compared to the control cell is indicative ofclostridial toxin activity. In another embodiment, a method of theinvention includes the step of detecting acceptor fluorescence intensityof the contacted cell relative to a control cell, where decreasedacceptor fluorescence intensity of the contacted cell as compared to thecontrol cell is indicative of clostridial toxin activity. In a furtherembodiment, a method of the invention includes the step of detecting anacceptor emission maximum and a donor fluorophore emission maximum ofthe contacted cell relative to a control cell, where a shift in emissionmaxima from near the acceptor emission maximum to near the donorfluorophore emission maximum is indicative of clostridial toxinactivity. In yet a further embodiment, a method of the inventionincludes the step of detecting the ratio of fluorescence amplitudes nearan acceptor emission maximum to the fluorescence amplitudes near a donorfluorophore emission maximum of the contacted cell relative to a controlcell, where a decreased ratio in the contacted cell as compared to thecontrol cell is indicative of clostridial toxin activity. In stillanother embodiment, a method of the invention includes the step ofdetecting the excited state lifetime of the donor fluorophore in thecontacted cell relative to a control cell, where an increased donorfluorophore excited state lifetime in the contacted cell as compared tothe control cell is indicative of clostridial toxin activity. Ifdesired, the step of determining resonance energy transfer can berepeated at one or more later time intervals. In addition, conditionssuitable for clostridial toxin activity can be optionally selected suchthat the assay is linear.

As discussed further below, it is understood that the methods of theinvention are applicable to crude samples as well as highly purifieddichain toxins. As non-limiting examples, a method of the invention canbe useful to assay for clostridial toxin activity in a food or beveragesample; to assay a sample from a human or animal, for example, exposedto a clostridial toxin or having one or more symptoms of a clostridialtoxin; to follow activity during production and purification ofclostridial toxin, and to assay formulated clostridial toxin products,including pharmaceuticals and cosmetics.

A method of the invention can be used to determine the activity of anyclostridial toxin. In one embodiment, a method of the invention relieson a cell containing a BoNT/A substrate to determine BoNT/A activity.Such a BoNT/A substrate can be any of the BoNT/A substrates describedherein, for example, a BoNT/A substrate containing at least sixconsecutive residues of SNAP-25, where the six consecutive residuesinclude Gln-Arg. In another embodiment, a method of the invention relieson a cell containing a BoNT/B substrate to determine BoNT/B activity.Such a BoNT/B substrate can be any of the BoNT/B substrates describedherein, for example, a BoNT/B substrate containing at least sixconsecutive residues of VAMP, where the six consecutive residues includeGln-Phe. A method of the invention also can utilize a cell containing aBoNT/C1 substrate to determine BoNT/C1 activity. A BoNT/C1 substrateuseful in a method of the invention can be any of the BoNT/C1 substratesdescribed herein, for example, a BoNT/C1 substrate containing at leastsix consecutive residues of syntaxin, where the six consecutive residuesinclude Lys-Ala, or containing at least six consecutive residues ofSNAP-25, where the six consecutive residues include Arg-Ala.

In another embodiment, a method of the invention relies on a cellcontaining a BoNT/D substrate to determine BoNT/D activity. Such aBoNT/D substrate can be any of the BoNT/D substrates described herein,for example, a BoNT/D substrate containing at least six consecutiveresidues of VAMP, where the six consecutive residues include Lys-Leu. Ina further embodiment, a method of the invention is practiced with a cellcontaining a BoNT/E substrate to determine BoNT/E activity. A BoNT/Esubstrate useful in a method of the invention can be any of the BoNT/Esubstrates described herein, for example, a BoNT/E substrate containingat least six consecutive residues of SNAP-25, where the six consecutiveresidues include Arg-Ile. In yet a further embodiment, a method of theinvention relies on a cell containing a BoNT/F substrate to determineBoNT/F activity. A BoNT/F substrate useful in a method of the inventioncan be any of the BoNT/F substrates described herein, for example, aBoNT/F substrate containing at least six consecutive residues of VAMP,where the six consecutive residues include Gln-Lys.

A method of the invention also can utilize a cell containing a BoNT/Gsubstrate to determine BoNT/G activity. A BoNT/G substrate useful in amethod of the invention can be any of the BoNT/G substrates describedherein, for example, a BoNT/G substrate containing at least sixconsecutive residues of VAMP, where the six consecutive residues includeAla-Ala. A method of the invention also can be useful to determine TeNTprotease activity and, in this case, relies on a cell containing a TeNTsubstrate. Any of the TeNT substrates described herein can be useful ina method of the invention, for example, a TeNT substrate containing atleast six consecutive residues of VAMP, where the six consecutiveresidues include Gln-Phe.

A variety of samples are useful in the methods of the invention. As usedherein, the term “sample” means any biological matter that contains orpotentially contains an active clostridial toxin. Thus, the term sampleencompasses but is not limited to purified or partially purifiedclostridial toxin; recombinant single chain or dichain toxin with anaturally or non-naturally occurring sequence; recombinant clostridialtoxin with a modified protease specificity; recombinant clostridialtoxin with an altered cell specificity; chimeric toxin containingstructural elements from multiple clostridial toxin species or subtypes;bulk toxin; formulated product; cells or crude, fractionated orpartially purified cell lysates, for example, engineered to include arecombinant nucleic acid encoding a clostridial toxin; bacterial,baculoviral and yeast lysates; raw, cooked, partially cooked orprocessed foods; beverages; animal feed; soil samples; water samples;pond sediments; lotions; cosmetics; and clinical formulations. Itfurther is understood that the term sample encompasses tissue samples,including, without limitation, mammalian tissue samples, livestocktissue samples such as sheep, cow and pig tissue samples; primate tissuesamples; and human tissue samples. Such samples encompass, withoutlimitation, intestinal samples such as infant intestinal samples, andtissue samples obtained from a wound.

The concentration of purified or partially purified clostridial toxinassayed in a method of the invention generally is in the range of about0.0001 to 5000 ng/ml toxin, for example, about 0.001 to 5000 ng/ml, 0.01to 5000 ng/ml, 0.1 to 5000 ng/ml, 1 to 5000 ng/ml, or 10 to 5000 ng/mltoxin, which can be, for example, purified recombinant dichain toxin orformulated clostridial toxin product containing human serum albumin andexcipients. Generally, the amount of purified toxin assayed in a methodof the invention is in the range of 0.1 pg to 10 μg. It is understoodthat purified, partially purified or crude samples can be diluted suchthat the sample is within a convenient range for assaying forclostridial toxin activity against a standard curve. Similarly, oneskilled in the art understands that a sample can be diluted or theamount of sample otherwise limited, such that the assay is linear since,at increasingly high concentrations of toxin, linearity of the assay canbe sacrificed.

One skilled in the art understands that, in a method of the invention, acell can be contacted with clostridial toxin for any of a variety oflengths of time depending, in part, on the type of cell used, theaffinity of the toxin receptor expressed and the amount and type oftoxin assayed. As non-limiting examples, the cell can be contacted withtoxin for up to one hour, two hours, four hours, eight hours, sixteenhours, 24 hours, 48 hours or 72 hours.

In a method of the invention, resonance energy transfer can bedetermined by a variety of means. In one embodiment, the step ofdetermining resonance energy transfer includes detecting donorfluorescence intensity of the contacted cell, where increased donorfluorescence intensity of the contacted cell as compared to the controlcell is indicative of clostridial toxin activity. In another embodiment,the step of determining resonance energy transfer includes detectingacceptor fluorescence intensity of the contacted cell, where decreasedacceptor fluorescence intensity of the contacted cell as compared to thecontrol cell is indicative of clostridial toxin activity. In a furtherembodiment, the step of determining resonance energy transfer includesdetecting the acceptor emission maximum and the donor fluorophoreemission maximum, where a shift in emission maxima from near an acceptoremission maximum to near a donor fluorophore emission maximum isindicative of clostridial toxin activity. In an additional embodiment,the step of determining resonance energy transfer includes detecting theratio of fluorescence amplitudes near an acceptor emission maximum tofluorescence amplitudes near a donor fluorophore emission maximum, wherea decreased ratio in the contacted cell as compared to the control cellis indicative of clostridial toxin activity. In yet a furtherembodiment, the step of determining resonance energy transfer ispracticed by detecting the excited state lifetime of the donorfluorophore in the contacted cell, where an increased donor fluorophoreexcited state lifetime in the contacted cell as compared to the controlcell is indicative of clostridial toxin activity.

In a method of the invention for determining clostridial toxin activity,a cell is contacted with a sample, the cell containing a clostridialtoxin substrate that includes a first donor fluorophore, a firstacceptor having an absorbance spectrum which overlaps the emissionspectrum of the donor fluorophore, and a first clostridial toxinrecognition sequence containing a cleavage site, where the cleavage siteintervenes between the donor fluorophore and the acceptor and where,under the appropriate conditions, resonance energy transfer is exhibitedbetween the donor fluorophore and the acceptor. If desired, a secondclostridial toxin substrate can be included in the same cell; thissecond substrate contains a second donor fluorophore and second acceptorhaving an absorbance spectrum which overlaps the emission spectrum ofthe second donor fluorophore, and a second clostridial toxin recognitionsequence that is cleaved by a different clostridial toxin than the toxinthat cleaves the first clostridial toxin recognition sequence. The donorfluorophore-acceptor pair in the second substrate can be the same ordifferent from the donor fluorophore-acceptor pair in the firstsubstrate. In this way, a single sample conveniently can be assayed forthe presence of more than one clostridial toxin.

It is understood that one can assay for any combination of clostridialtoxins, for example, two, three, four, five, six, seven, eight, or moreclostridial toxins. One can assay, for example, any combination of two,three, four, five, six, seven or eight of BoNT/A, BoNT/B, BoNT/C1,BoNT/D, BoNT/E, BoNT/F, BoNT/G and TeNT. As an example, seven cells ofthe same of different types, selected as described hereinabove, whichcontain seven substrates, each of which includes fluorescein andtetramethylrhodamine flanking a BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E,BoNT/F or BoNT/G recognition sequence and cleavage site. These cellseach are contacted with a sample under conditions suitable for botulinumtoxin activity before exciting the donor fluorescein at an absorptionwavelength of about 488 nm and determining energy transfer. A shift inthe emission maximum of the acceptor, tetramethylrhodamine (585 nm) tothat of fluorescein (520 nm) is indicative of activity of at least onebotulinum toxin. Such an assay can be useful, for example, for assayingfood samples or tissue samples for the presence of any botulinum orother clostridial toxin and can be combined, if desired, with one ormore subsequent assays for individual clostridial toxins or specificcombinations of clostridial toxins.

In another embodiment, a single sample is assayed for two or moredifferent clostridial toxins using two or more different clostridialtoxin substrates contained in the same or different cells such asneurons, with each substrate containing a different donorfluorophore-acceptor pair. The use of multiple substrates can be usefulfor extending the dynamic range of an assay, as described, for example,in U.S. Pat. No. 6,180,340. As an example of the use of multipleclostridial toxin substrates, a single sample can be assayed for BoNT/Aand BoNT/B activity using a cell containing first and second clostridialtoxin substrates: the first clostridial toxin substrate contains thedonor fluorophore fluorescein and the acceptor tetramethylrhodamine withan intervening BoNT/A recognition sequence and cleavage site, and thesecond clostridial toxin substrate contains the donor fluorophore EDANSand the acceptor DABCYL with an intervening BoNT/B recognition sequenceand cleavage site. The first donor fluorophore, fluorescein, is excitedat about 488 nm, and energy transfer is determined, with an increase inthe fluorescence intensity of fluorescein (at about 520 nm) indicativeof BoNT/A activity. The second donor fluorophore, EDANS, is excited atan absorption wavelength of about 340 nm, with an increase in thefluorescence intensity of EDANS (at about 490 nm) indicative of BoNT/Bactivity. Similarly, where two or more different donor fluorophores areto be used together to assay a single sample, one can combine, forexample, any combination or all of the following lanthanides: terbium,dysprosium, europium and samarium (EG&G-Wallac). These lanthanides havespectra that are clearly distinguishable on the basis of decay time andwavelength. Those skilled in the art understand that the first donorfluorophore can be excited before, at the same time, or after excitationof the second donor fluorophore, and that energy transfer of the firstsubstrate can be determined before, at the same time, or afterdetermining energy transfer of the second substrate.

The methods of the invention involve exciting a donor fluorophore whichis incorporated into a clostridial toxin substrate within a cell. Oneskilled in the art understands that a donor fluorophore generally isexcited at or near the optimal absorption wavelength (excitationwavelength) of the donor fluorophore. As an example, where the donorfluorophore is fluorescein, the donor can be excited, for example, at ornear the optimal absorption wavelength of 488 nm.

Proteolysis of the clostridial toxin substrate, and hence clostridialtoxin activity, can be detected by a variety of means, for example, bydetecting increased donor fluorescence intensity; decreased acceptorfluorescence intensity; a shift in emission maxima from near theacceptor emission maximum to near the donor fluorophore emissionmaximum; a decreased ratio of fluorescence amplitudes near the acceptoremission maximum to the fluorescence amplitudes near the donorfluorophore emission maximum; or an increased donor fluorophore excitedstate lifetime. It is understood that the relevant fluorescenceintensities or excited state lifetimes are detected at the appropriatewavelength or range of wavelengths. As an example, where donorfluorescence intensity is detected, the appropriate wavelength is at ornear the emission maxima of the donor fluorophore, or is a range ofwavelengths encompassing or near to the emission maxima of the donorfluorophore.

It is recognized that changes in the absolute amount of clostridialtoxin substrate in the cell, excitation intensity, and turbidity orother background absorbance at the excitation wavelength effects thefluorescence intensities of donor and acceptor fluorophores roughly inparallel. Thus, it is understood that a ratio of emission intensities isindependent of the absolute amount of substrate, excitation intensity,and turbidity or other background absorbance, and can be a usefulindicator of clostridial toxin activity. Similarly, one skilled in theart understands that the excitation state lifetime of a donorfluorophore is independent of the absolute amount of substrate,excitation intensity, and turbidity or other background absorbance andcan be useful in a method of the invention.

In one embodiment, a method of the invention is practiced by detectingdonor fluorescence intensity, with increased donor fluorescenceintensity indicative of clostridial toxin activity. Such increasedintensity can be, for example, at least two-fold, three-fold, five-fold,ten-fold, twenty-fold or more relative to fluorescence intensity at thesame wavelength of the same or similar cell not contacted with sample.

For detection of donor fluorescence intensity, excitation is set at thewavelength of donor fluorophore absorption, and the emission of thedonor fluorophore is monitored. The emission wavelength of the donorfluorophore generally is selected such that little or no contributionfrom acceptor fluorescence is observed. The presence of acceptorquenches donor fluorescence. Energy transfer efficiency, E, iscalculated from E=1−I_(DA)/I_(D), where I_(DA) and I_(D) are donorintensities in the presence and absence of acceptor. Both are normalizedto the same donor fluorophore concentration. If desired, time resolvedmeasurements, for which donor fluorophore concentration is not required,can be performed using E=1−{T_(DA)}/T_(D), where {T_(DA)} and {T_(D)}are amplitude-averaged lifetimes of donor fluorophore in the presenceand absence of acceptor.

In one embodiment, the invention is practiced by detecting a shift inemission maxima from near the acceptor emission maximum to near thedonor fluorophore emission maximum as a determination of resonanceenergy transfer. As an example, where a tetramethylrhodamine acceptor iscombined with the donor fluorophore fluorescein, one can detect a shiftfrom predominantly red emission to predominantly green emission as anindicator of decreased resonance energy transfer and, therefore, ofclostridial toxin activity. It is understood that the observed shift inemission maxima generally will not be a complete shift but that onlypart of the emission intensity will be shifted to near the donorfluorophore emission maximum.

In several methods of the invention, resonance energy transfer of thecontacted cell is determined relative to a control cell. Such a controlcell generally is a cell of the same or similar type as the contactedcell and grown under the same conditions but which is not contacted withany sample or is contacted with a defined negative sample or a definedpositive sample. One skilled in the art understands that a variety ofcontrol cells are useful in the methods of the invention and that acontrol cell can be a positive control cell or a negative control cell.A control cell can be, for example, a negative control cell such as asimilar or identical cell containing the same or similar clostridialtoxin substrate that is contacted with a similar, defined negativesample, which is known to lack active clostridial toxin, or that is notcontacted with any sample. A control cell also can be, for example, apositive control cell such as a cell containing one or both cleavageproducts that result from proteolysis of the clostridial toxin substrateat the cleavage site or a cell containing the same or similar substratecontacted with a defined positive sample, which is known to includeactive clostridial toxin. Positive control cells include cellscontaining the donor fluorophore-containing cleavage product, cellscontaining the acceptor-containing cleavage product, and cellscontaining both cleavage products.

The methods of the invention for determining clostridial toxin activityinvolve determining resonance energy transfer of a cell containing aclostridial toxin substrate contacted with a sample relative to acontrol cell and can be practiced as “fixed-time” assays or ascontinuous time assays. Thus, in one embodiment, the FRET determinationis repeated at one or more later time intervals. Fluorescence resonanceenergy transfer can be determined, for example, at two or more, five ormore, ten or more, or twenty or more different times. Fluorescenceintensities and other indicators of FRET also can be detectedcontinuously by well known methods (see, for example, Wang et al.,supra, 1993; Holskin et al., supra, 1995; and Kakiuchi et al., supra,1999).

In a method of the invention, fluorescence of a contacted cell typicallyis determined using a fluorimeter. In general, excitation radiation froman excitation source having a first wavelength passes through excitationoptics. The excitation optics cause the excitation radiation to excitethe substrate in the cell. In response, fluorophores in the substrateemit radiation which has a wavelength that is different from theexcitation wavelength. Collection optics then collect the emission; ifdesired, the device includes a temperature controller to maintain thecell at a specific temperature while being scanned. If desired, amulti-axis translation stage moves a microtiter plate containing aplurality of samples in order to position different wells to be exposed.It is understood that the multi-axis translation stage, temperaturecontroller, auto-focusing feature, and electronics associated withimaging and data collection can be managed by the appropriate digitalcomputer.

It is further understood that the methods of the invention can beautomated and can be configured in a high-throughput or ultrahigh-throughput format using, without limitation, 96-well, 384-well or1536-well plates. As one example, fluorescence emission can be detectedusing the Molecular Devices FLIPR-instrumentation system (MolecularDevices; Sunnyvale, Calif.), which is designed for 96-well plate assays(Schroeder et al., J. Biomol. Screening 1:75-80 (1996)). FLIPR utilizesa water-cooled 488 nm argon ion laser (5 watt) or a xenon arc lamp and asemiconfocal optimal system with a charge-coupled device (CCD) camera toilluminate and image the entire plate. The FPM-2 96-well plate reader(Folley Consulting and Research; Round Lake, Ill.) also can be useful indetecting fluorescence emission in the methods of the invention. Oneskilled in the art understands that these and other automated systemswith the appropriate spectroscopic compatibility such as the ECLIPSEcuvette reader (Varian-Cary; Walnut Creek, Calif.), the SPECTRA_(max)GEMINI XS (Molecular Devices) and other systems from, for example,Perkin Elmer can be useful in the methods of the invention.

The methods of the invention can be practiced, if desired, in vivo; asnon-limiting examples, the methods of the invention can be practiced ina mouse, rat, worm or fish. An in vivo method of the invention fordetermining clostridial toxin activity can be practiced by (a)administering to an animal a substrate composition containing a deliveryagent and a clostridial toxin substrate that includes a donorfluorophore; an acceptor having an absorbance spectrum overlapping theemission spectrum of the donor fluorophore; and a clostridial toxinrecognition sequence containing a cleavage site that intervenes betweenthe donor fluorophore and the acceptor, where resonance energy transferis exhibited between the donor fluorophore and the acceptor under theappropriate conditions; (b) treating the animal with a sample; (c)exciting the donor fluorophore; and (d) determining resonance energytransfer of the treated animal relative to a control animal, where adifference in resonance energy transfer of the treated animal ascompared to the control animal is indicative of clostridial toxinactivity. As an example, an HIV TAT based delivery agent has been shownto efficiently deliver a target protein in vivo (Schwartz et al.,Science 285:1569-1572 (1999)). In one embodiment, an in vivo method ofthe invention is practiced with a substrate composition which includesan HIV TAT derived delivery agent. In a further embodiment, thesubstrate composition is administered into the spinal cord of theanimal. In yet a further embodiment, the persistence of toxin activityover time is assayed by determining resonance energy transfer two ormore times.

The present invention relies, in part, on FRET which is a physicalprocess whereby energy is transferred non-radiatively from an exciteddonor fluorophore to an acceptor, which may be another fluorophore,through intramolecular long-range dipole-dipole coupling. FRET isdependent on the inverse sixth power of the intramolecular separation ofthe donor fluorophore and acceptor, and for effective transfer, thedonor fluorophore and acceptor are in close proximity, separated, forexample, by about 10 A to about 100 A. Effective energy transfer isdependent on the spectral characteristics of the donor fluorophore andacceptor as well as their relative orientation. For effective transferover 10 to 100 A, the quantum yield of the donor fluorophore generallyis at least 0.1, and the absorption coefficient of the acceptorgenerally is at least 1000 (see Clegg, Current Opinion in Biotech.6:103-110 (1995); and Selvin, Nature Structural Biol. 7:730-734 (2000)).

In a clostridial toxin substrate useful in the invention, the donorfluorophore and acceptor are selected so that the donor fluorophore andacceptor exhibit resonance energy transfer when the donor fluorophore isexcited. One factor to be considered in choosing the donorfluorophore/acceptor pair is the efficiency of FRET between the donorfluorophore and acceptor. In one embodiment, the invention relies on aclostridial toxin substrate in which, under optimal conditions, theefficiency of FRET between the donor fluorophore and acceptor is atleast 10%. In another embodiment, the invention relies on a clostridialtoxin substrate in which, under optimal conditions, the efficiency ofFRET between the donor fluorophore and acceptor is at least 20%. Instill further embodiments, the invention relies on a clostridial toxinsubstrate in which, under optimal conditions, the efficiency of FRETbetween the donor fluorophore and acceptor is at least 30%, 40%, 50%,60%, 70% or 80%.

As is well known in the art, the efficiency of FRET is dependent on theseparation distance and the orientation of the donor fluorophore andacceptor as described by the Förster equation, as well as thefluorescent quantum yield of the donor fluorophore and the energeticoverlap with the acceptor. In particular, the efficiency (E) of FRET canbe determined as follows:E=1−F _(DA) /F _(D)=1/(1+(R/R ₀)⁶)

-   -   where F_(DA) and F_(D) are the fluorescence intensities of the        donor fluorophore in the presence and absence of the acceptor,        respectively, and R is the distance between the donor        fluorophore and the acceptor.

The Förster radius (R_(o)) is the distance at which resonance energytransfer is 50% efficient, that is, 50% of excited donor fluorophoresare deactivated by FRET. The magnitude of the Förster radius depends onthe quantum yield of the donor fluorophore; the extinction coefficientof the acceptor; and the overlap between the donor fluorophore'semission spectrum and the acceptor's excitation spectrum.R _(o)=[8.8×10²³ Xκ ² Xn ⁻⁴ XQY _(D) XJ(λ)]^(1/6)Δ

-   -   where κ²=dipole orientation factor (range 0 to 4; κ²=⅔ for        randomly oriented donors and acceptors)        ${QY}_{D} = \begin{matrix}        {{fluorescence}{\quad\quad}{quantum}\quad{yield}\quad{of}\quad{the}\quad{donor}} \\        {{in}\quad{the}\quad{absence}\quad{of}\quad{the}\quad{acceptor}}        \end{matrix}$ n = refractive  index $\begin{matrix}        {{J(\lambda)} = {{spectral}\quad{overlap}\quad{integral}}} \\        {= {❘{{ɛ_{.A}(\lambda)}{{XF}_{D}(\lambda)}X\quad\lambda^{4}d\quad\lambda\quad{cm}^{3}M^{- 1}}}}        \end{matrix}$    -   where ε_(.A)=extinction coefficient of acceptor    -   F_(D)=fluorescence emission intensity of donor as a fraction of        the total integrated intensity (Förster, Ann. Physik 2:55-75        (1948)).

Typical Förster radius values for various donor fluorophore/acceptorpairs are given in Table B below (see, also, Wu and Brand, AnalyticalBiochem. 218:1-13 (1994)). Comprehensive lists of Förster radii also areknown in the art (see, for example, Berlman, Energy Transfer Parametersof Aromatic Compounds Academic Press, New York 1973). Furthermore, thoseskilled in the art recognize that component factors of the Försterradius (R_(o)) are dependent upon the environment such that the actualvalue observed can vary from the listed value.

Any of a number of donor fluorophores and acceptors in variouscombinations can be included in a clostridial toxin substrate useful inthe invention. A donor fluorophore generally is selected such that thereis substantial spectral overlap between the emission spectrum of thedonor fluorophore and the excitation spectrum of the acceptor. Inaddition, a donor fluorophore can be selected, for example, to have anexcitation maximum near a convenient laser frequency such asHelium-Cadmium 442 nm or argon 488 nm, since laser light serves as aconvenient and effective means to excite the donor fluorophore. In oneembodiment, the wavelength maximum of the emission spectrum of theacceptor moiety is at least 10 nm greater than the wavelength maximum ofthe excitation spectrum of the donor fluorophore. In a furtherembodiment, the acceptor is a fluorophore having an emission spectrum inthe red portion of the visible spectrum. In an additional embodiment,the acceptor is a fluorophore having an emission spectrum in theinfrared region of the spectrum. A variety of donor fluorophore-acceptorpairs, and their Förster radii, are provided herein in Tables B and C.See, also, Haugland, Handbook of Fluorescent Probes and ResearchChemicals 6^(th) Edition, Molecular Probes, Inc., Eugene, Oreg., 1996.TABLE B Exemplary Donor Fluorophores and Acceptors Donor fluorophoreAcceptor Ro (Δ) Reference Fluorescein TMR 49-54 Johnson et al.,Biochemistry 32:6402-6410 (1993); Odom et al., Biochemistry 23:5069-5076(1984) Fluorescein QSY ® 7 61 — EDANS DABCYL 33 — Napthalene Dansyl 22Haas et al., Proc. Natl. Acad. Sci. USA 72:1807- 1811 (1975) IANBD DDPM25 Kasprzyk et al., Biochemistry 22:1877-1882 (1983) IAEDANS DDPM 25-29Dalbey et al., Biochemistry 22:4696-4706 (1983); Cheung et al., Biophys.Chem. 40:1-17 (1991) DNSM LY 26-32 Nalin et al., Biochemistry28:2318-2324 (1985) IAEDANS IANBD 27-51 Franzen et al., Biochemistry19:6080-6089 (1980); First et al., Biochemistry 28:3606- 3613 (1989) ε-A_(F2)DNB 29 Perkins et al., J. Biol. Chem. 259:8786-8793 (1984) PyreneBimane 30 Borochov-Neori and Montal, Biochemistry 28:1711-1718 (1989)ANAI IPM 30 Peerce and Wright, Proc. Natl. Acad. Sci. USA 83:8092-8096(1986) IAANS IAF 31 Grossman, Biochim. Biophys. Acta 1040:276-280 (1990)ε-A F₂DPS 31 Perkins et al., supra, 1984 ε-A DDPM 31 Miki and Mihashi,Biochim. Biophys. Acta 533:163-172 (1978) IAEDANS TNP 31-40 Takashi etal., Biochemistry 21:5661-5668 (1982); dos Remedios and Cooke, Biochim.Biophys. Acta 788:193-205 (1984) MNA DACM 32 Amir and Haas, Biochemistry26:2162-2175 (1987) PM NBD 32 Snyder and Hammes, Biochemistry 24:2324-2331 (1985) FITC TNP-ATP 32 Amler et al., Biophys. J. 61:553-568 (1992)DANZ DABM 34 Albaugh and Steiner, J. Phys. Chem. 93:8013- 8016 (1989)NCP CPM 34 Mitra and Hammes, Biochemistry 28:3063-3069 (1989) NAA DNP33-37 McWherter et al., Biochemistry 25:1951-1963 (1986) LY TNP-ATP 35Nalin, supra, 1985 IAF dil-C₁₈ 35 Shahrokh et al., J. Biol. Chem.266:12082-12089 (1991) IAF TMR 37 Taylor et al., J. Cell Biol.89:362-367 (1981) FMA FMA 37 Dissing et al., Biochim. Biophys. Acta553:66-83 (1979) PM DMAMS 38 Lin and Dowben, J. Biol. Chem.258:5142-5150 (1983) mBBR FITC 38 Tompa and Batke, Biochem. Int.20:487-494 (1990) mBBR DABM 38 Kasprzak et al., Biochemistry27:4512-4523 (1988) ε-A NBD 38 Miki and Lio, Biochim. Biophys. Acta790:201-207 (1984) Pyrene Coumarin 39 Borochov-Neori and Montal, supra,1989 IPM FNAI 39 Peerce and Wright, supra, 1986 IAEDANS DABM 40 Tao etal. Biochemistry 22:3059-3066 (1983) IAEDANS TNP-ATP 40 Tao et al.,supra, 1983 ε-A IANBD 40 Miki and Wahl, Biochim. Biophys. Acta 786:188-196 (1984) NBD SRH 40-74 Wolf et al., Biochemistry 31:2865-2873 (1992)ISA TNP 42 Jacobson and Colman, Biochemistry 23:3789- 3799 (1984) DansylODR 43 Lu et al., J. Biol. Chem. 264:12956-12962 (1989) DANZ IAF 44-49Cheung et al., Biochemistry 2l:5135-5142 (1983) FNAI EITC 45 Peerce andWright, supra, 1986 NBD LRH 45-70 Wolf et al., supra, 1992 IAF EIA 46Taylor et al., supra, 1981 FITC ENAI 46 Peerce and Wright, supra, 1986Proflavin ETSC 46 Robbins et al., Biochemistry 20:5301-5309 (1981) CPMTNP-ATP 46 Snyder and Hammes, supra, 1985 IAEDANS IAF 46-56 Franzen,supra, 1985; Grossman, supra, 1990 CPM Fluorescein 47 Thielen et al.,Biochemistry 23:6668-6674 (1984) IAEDANS FITC 49 Jona et al., Biochim.Biophys. Acta 1028:183-199 (1990); Birmachu et al., Biochemistry28:3940- 3947 (1989) IAF TMR 50 Shahrokh et al., J. Biol. Chem.266:12082-12089 (1991) CF TR 51 Johnson et al., supra, 1993 CPM TRS 51Odom et al., supra, 1984 ε-A TNP-ATP 51 dos Remedios and Cooke, supra,1984 CPM FM 52 Odom et al., supra, 1984 LY EM 53 Shapiro et al., J.Biol. Chem. 266:17276-17285 (1991) FITC EITC 54 Carraway et al., J.Biol. Chem. 264:8699-8707 (1989) IAEDANS DiO-C₁₄ 57 Shahrokh et al.,supra, 1991 IAF ErITC 58 Amler et al., supra, 1992 FITC EM 60Kosk-Kosicka et al., J. Biol. Chem. 264:19495- 19499 (1989) FITC ETSC61-64 Robbins et al., supra, 1981 FITC ErITC 62 Amler et al., supra,1992 BPE CY5 72 Ozinskas et al., Anal. Biochem. 213:264-270 (1993)Fluorescein Fluorescein 44 — BODIBY FL ® BODIPY FL ® 57 —ANAI, 2-anthracence N-acetylimidazole;BPE, B-phycoerythrin;CF, carboxyfluorescein succinimidyl ester;CPM, 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin;CY5, carboxymethylindocyanine-N-hydroxysuccinimidyl ester;dil-C₁₈, 1,1′-dioctadecyl-3-3,3,3′,3′-tetramethyl-indocarbocyanine;diO-C₁₄, 3,3′-ditetradecyloxacarbocyanine;DABM, 4-dimethylaminophenylazo-phenyl-4′-maleimide;DACM, (7-(dimethylamino)coumarin-4-yl)-acetyl;DANZ, dansylaziridine; DDPM,N-(4-dimethylamino-3,5-dinitrophenyl)maleimide;DMAMS, dimethylamino-4-maleimidostilbene;DSMN, N-(2,5′-dimethoxystiben-4-yl)-maleimide;DNP, 2,4-dinitrophneyl;ε-A, 1,N⁶-ethenoadenosine;EIA, 5-(iodoacetetamido)eosin;EITC, eosin-5-isothiocyanate;ENAI, eosin N-acETYLIMIDAZOLE;EM, eosin maleimide;ErlTC, erythrosin-5′-isothiocyanate;ETSC, eosin thiosemicarazide;F₂DNB, 1,5-difluro-2,4′-dinitrobenzene;F₂DPS, 4,4′-difluoro-3,3′-dinitrophenylsulfone;FITC, fluorescein thiosemicarbazide;IAANS, 2-(4′-iodoacetamido)anilino)napthalene-6-sulfonic acid;IAEDANS, 5-(2-((iodoacetyl)amino)ethyl)amino)-napthlene-1-sulfonic acid;IAF, 5-iodoacetamidofluorescein;IANBD,N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole;IPM, 3(4-isothiocyanatophenyl)7-diethyl-4-amino-4-methylcoumarin;ISA, 4-(iodoacetamido)salicylic acid;LRH, lissaminerhodamine;LY, Lucifer yellow;mBBR, monobromobiamane;MNA, (2-methoxy-1-naphthyl)-methyl;NAA, 2-napthoxyacetic acid;NBD, 7-nirto-2,1,3-benzoxadiazol-4-yl;NCP, N-cyclohexyl-N′-(1-pyrenyl)carbodiimide;ODR, octadecylrhodamine;PM, N-(1-pyrene)-maleimide;SRH, sulforhodamine;TMR, tetramethylrhodamine;TNP, trinitrophenyl; andTR, Texas Red

An aromatic amino acid such as tryptophan or tyrosine also can be adonor fluorophore useful in a clostridial toxin substrate and thecompositions and methods disclosed herein. Exemplary donorfluorophore-acceptor pairs in which tryptophan or tyrosine is the donorfluorophore and relevant Förster distances are shown in Table C below.Modified amino acids also can be useful as donor fluorophores oracceptors in a clostridial toxin substrate useful in the invention. Suchfluorescent or quenching modified amino acids are known in the art andinclude, for example, the fluorescent amino acid L-pyrenylalanine (Pya)and the non-fluorescent acceptor p-nitrophenylalanine (Nop), asdescribed, for example, in Anne et al., Analytical Biochem. 291:253-261(2001). TABLE C Föorster Distances Using Trp as a Donor Donor AcceptorR_(O) (Δ) Reference Trp Ru(III)(NH3)5 12-16 Recchia et al., Biochim.Biophys. Acta 702:105-111 (1982) Trp Nitrobenzoyl 16 Wiczk et al., J.Fluo 1:273-286 (1991) Trp Dansyl 21 Steinberg, Annu. Rev. Biochem.40:83-114 (1971) Trp IAEDANS 22 Matsumoto and Hammes, Biochemistry14:214-224 (1975) Trp ANS 23 Conrad and Brand, Biochemistry 7:777-787(1968) Trp Anthroyloxy 24 Wiczk et al., supra, 1991 Trp TNB 24 Wu andBrand, Biochemistry 31:7939-7947 (1992) Trp Anthroyl 25 Burgun et al.,Arch. Biochem. Biophys. 286:394-401 (1991) Trp Tyr-NO₂ 26 Steiner etal., J. Fluo. 1:15-22 (1991) Trp Pyrene 28 Vekshin, Mol. Biol.17:827-832 (1983) Trp Heme 29 Ladokhin et al., Proc. SPIE 1640:562-569(1992) Trp NBS 30 Wiczk et al., supra, 1991 Trp DNBS 33 Wiczk et al.,supra, 1991 Trp DPH 40 Le Doan et al., Biochim. Biophys. Acta735:259-270 (1983)

In view of the above, it is understood that a variety of donorfluorophore/acceptor pairs can be included in a clostridial toxinsubstrate useful in the invention. A donor fluorophore-acceptor pairuseful in a clostridial toxin substrate can be, for example, the donorfluorophore fluorescein in combination with rhodamine; texas red; eosin;ROX (6-carboxy-X-rhodamine; Applied Biosystems Division of Perkin-ElmerCorporation; Foster City, Calif.); or TAMRA(N,N,N′,N′-tetramethyl-6-carboxy-rhodamine; Applied Biosystems). A donorfluorophore-acceptor pair useful in the invention also can be, forexample, the donor fluorophore cascade blue with fluorescein as anacceptor; the donor fluorophore BODIPY®-530/550(4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-S-indacene) in combinationwith BODIPY®-542/563(4,4-difluoro-5-p-methoxyphenyl-4-bora-3a,4a-diaza-S-indacene) as anacceptor; or BODIPY®-542/563 in combination with BODIPY®-564/570(4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-S-indacene) as an acceptor. Itis understood that the numbers following the name BODIPY®-reflect theexcitation and emission maxima of the molecule; BODIPY®-compounds arecommercially available from Molecular Probes (Eugene, Oreg.).

Clostridial toxin substrates useful in the invention include those inwhich the donor fluorophore is fluorescein. In one embodiment, theclostridial toxin substrate includes fluorescein as the donorfluorophore and tetramethylrhodamine as the acceptor. Such a substratecan be excited in the range of 480 to 505 nm, for example, at 488 nm or492 nm, and emission detected at 520 nm (λ_(em) fluorescein), 585 nm(λ_(em) tetramethylrhodamine), or both. Prior to cleavage of thesubstrate at the clostridial toxin cleavage site, thetetramethylrhodamine emission intensity is greater than that offluorescein; substrate cleavage results in a change in the ratio offluorescein to tetramethylrhodamine intensity. Cleavage generallyresults in fluorescein becoming the dominant emitting fluorophore.Methods for preparing proteins and peptides containing fluorescein andtetramethylrhodamine are well known in the art (see, for example,Matsumoto et al., Bioorg. Med. Chem. Letters 10:1857-1861 (2000)).

A donor fluorophore useful in a clostridial toxin substrate also can be,for example, EDANS (λ_(Ab) 340 nM, λ_(Em) 490 nm), which can be combinedwith an acceptor such as DABCYL. Where DABCYL and EDANS are combined ina clostridial toxin substrate, energy is transferred from the EDANSdonor fluorophore to the DABCYL acceptor in the intact substrate,resulting in quenching of EDANS emission fluorescence. Upon cleavage atthe toxin cleavage site, fluorescence of the cleaved EDANS product isincreased and can be restored, for example, to the free donorfluorophore level. Efficient fluorescence quenching in the intactsubstrate occurs as a result of favorable energetic overlap of the EDANSemission spectrum and the DABCYL absorbance spectrum, and the relativelylong excited state lifetime of the EDANS donor fluorophore (Wang et al.,Tetrahedron Lett. 31:6493-6496 (1991); Holskin et al., Anal. Biochem.226:148-155 (1995); and Wang et al., Anal. Biochem. 210:351-359 (1993)).

Dansyl (DNS or 5-dimethylaminonaphthalene-1-sulfonyl) also can be auseful as a donor fluorophore or acceptor in a clostridial toxinsubstrate. In one embodiment, the clostridial toxin substrate includesdansyl as the donor fluorophore; a dansyl donor can be combined, forexample, with a nitrophenyl residue acceptor such as Phe(pNO2), whichacts as a quencher when in proximity to the dansyl donor fluorophore.Substrates containing a dansyl donor fluorophore, for example, incombination with a nitrophenyl residue can be prepared as described, forexample, in Florentin et al., Anal. Biochem. 141:62-69 (1984), orGoudreau et al., Anal. Biochem. 219:87-95 (1994). In another embodiment,the clostridial toxin substrate contains dansyl as the acceptor. Adansyl acceptor can act as a quencher when combined, for example, with adonor fluorophore such as Trp (λ_(ex) 290 nm, λ_(em) 360 nm). In aclostridial toxin substrate containing Trp and dansyl, Trp fluorescencecan be quenched 60% by energy transfer to the dansyl group, and thisquenching can be significantly reduced or abolished in the presence oftoxin protease activity at the toxin cleavage site (see, for example,Geoghegan et al., FEBS Letters 262:119-122 (1990)).

It is understood that donor-acceptor pairs having well-separatedemission maxima can be useful in clostridial toxin substrates and,therefore, in the substrate compositions, cells and methods of theinvention. Well-separated emission maxima allow altered acceptoremission to be detected without donor emission contamination. A donorfluorophore, or acceptor, or both, can emit, for example, in thefar-red, for example, greater than 650 nm. Such far-red emitting donorfluorophores and acceptors include cyanine dyes such as Cy5, Cy5.5 andCy7 (Selvin, supra, 2000). In one embodiment, a clostridial toxinsubstrate includes Cy3 and Cy5 as the donor fluorophore-acceptor pair;Cy3 emits maximally at 570 nm, and Cy5 emits maximally at 670 nm. Suchcyanine dyes can be prepared by straightforward synthesis, as described,for example, in Gruber et al., Bioconj. Chem. 11:161-166 (2000).

A donor fluorophore useful in a clostridial toxin substrate also can be,for example, a lanthanide atom, also known as a rare-earth element.Lanthanides such as terbium (Tb), europium (Eu), dysprosium (Dy) andsamarium (Sm) have sharply spiked wavelengths, millisecond lifetimesfollowing an excitation pulse, are unpolarized, and have high quantumyields. A lanthanide donor fluorophore such as a terbium or europiumchelate can be combined with a variety of acceptors including organicdye acceptors. A Eu-chelate donor fluorophore can be combined, forexample, with allophycocyanin (APC), and a Tb-chelate donor fluorophorecan be combined, for example, with tetramethylrhodamine. Backgroundfluorescence due to direct excitation is eliminated temporally; thelifetimes of organic acceptors generally are in the nanosecond range,while the sensitized emission follows the lifetime of the donorfluorophore and is on the order of microseconds to milliseconds (seeSelvin, supra, 2000). Thus, determination of resonance energy transfercan be initiated relatively late following excitation, afternon-specific interfering fluorescence has faded away. Lanthanidechelates are well known in the art and are commercially available, forexample, from EG&G-Wallac (Turku, Finland).

A donor fluorophore useful in the invention also can be the well knownfluorophore (7-methoxycoumarin-4-yl) acetyl (Mca), which can be combinedwith an acceptor such as the quencher 2,4-dinitrophenyl (Dnp). See, forexample, Kakiuchi et al., J. Virol. Methods 80:77-84 (1999). When Mca iscombined with the appropriate quencher such as Dnp in a clostridialtoxin substrate, increased donor emission fluorescence from Mca (λ_(Em)393 nm) is detected upon cleavage at the clostridial toxin cleavage siteand is indicative of toxin activity.

A donor fluorophore useful in the invention also can be, for example, a2-aminobenzoyl (Abz) group, which can be combined, if desired, with aquencher such as 2,4-dinitrophenyl (Dnp). In an intact clostridial toxinsubstrate, the Dnp group quenches, by resonance energy transfer, thefluorescence of the Abz group; proteolytic cleavage of the substraterelieves quenching and results in an increase in fluorescenceproportional to the concentration of the released Abz fragment. Aclostridial toxin substrate containing, for example, Abz at theamino-terminus and a Dnp-derivatized residue such as lysine can beprepared by routine methods as described, for example, in Le Bonniec etal., Biochemistry 35:7114-7122 (1996)).

A donor fluorophore or acceptor useful in the invention also can be anAlexa Fluor®-dye, commercially available from Molecular Probes (Eugene,Oreg.). Alexa Fluor®-dyes useful in the substrate compositions, cellsand methods of the invention include, for example, Alexa Fluor-350,Alexa Fluor®-430, Alexa Fluor®-488, Alexa Fluor®-532, Alexa Fluor®-546,Alexa Fluor®-568, Alexa Fluor®-594, Alexa Fluor®-633, Alexa Fluor®-647,Alexa Fluor®-660 and Alexa Fluor®-680.

A donor fluorophore or acceptor useful in the invention also can be agenetically encoded dye (see above). It is understood that geneticallyencoded dyes such as GFP, BFP, CFP or YFP can form FRET pairs with eachother, or can be combined with other appropriate donor fluorophores oracceptors. In one embodiment, the clostridial toxin substrate includes agenetically encoded donor fluorophore and a genetically encodedacceptor.

In another embodiment, a clostridial toxin substrate includes afluorophore with a relatively long fluorescence lifetime of at least amicrosecond. Such an acceptor allows time-resolved measurement of thefluorescence emission due to the shorter fluorescence lifetimes ofimpurities and can enhance the signal to noise ratio. A useful donorfluorophore/acceptor pair for time-resolved fluorescence can be, forexample, a europium cryptate donor fluorophore such as Eu-trisbipyridinecryptate (TBP-EU³⁺, λ_(Ex) 337 nm) combined with the 105 kDaphycobiliprotein acceptor fluorophore, allophycocyanin (Sittampalam etal., Curr. Opin. Chem. Biol. 1:384-391 (1997)). The Eu-trisbipyridinecryptate has two bipyridyl groups that harvest light and channel it tothe caged EU³⁺; this donor fluorophore has a long fluorescence lifetimeand nonradiatively transfers energy to allophycocyanin when in closeproximity to the acceptor, exhibiting greater than 50% transferefficiency at a donor fluorophore-acceptor distance of 9.5 nm. BothTBP-EU³⁺ and allophycocyanin and their spectroscopic characteristics arevery stable in biological media, and allophycocyanin emits (λ_(Em)=665nm) with the long lifetime of the donor, allowing time-resolveddetection (Kolb et al., J. Biomol. Screening 1:203-210 (1996)). Methodsof preparing substrates containing such donor fluorophore-acceptor pairsare well known in the art as described, for example, in Kolb et al.,supra, 1996, and Sittampalam et al., supra, 1997.

In a further embodiment, the invention relies on a clostridial toxinsubstrate which contains a non-fluorescent acceptor, sometimesdesignated a “true quencher.” A non-fluorescent acceptor can be useful,for example, in eliminating background fluorescence resulting fromdirect (nonsensitized) acceptor excitation. A variety of non-fluorescentacceptors are known in the art including, for example, DABCYL and QSY-7®dyes (see Molecular Probes, supra, 1996).

A clostridial toxin substrate useful in the invention contains aclostridial toxin cleavage site which is positioned between a donorfluorophore and an acceptor. In one embodiment, the donor fluorophore ispositioned amino-terminal of the cleavage site while the acceptor ispositioned carboxy-terminal of the cleavage site. In another embodiment,the donor fluorophore is positioned carboxy-terminal of the cleavagesite while the acceptor is positioned amino-terminal of the cleavagesite.

One skilled in the art understands that there are several considerationsin selecting and positioning a donor fluorophore and acceptor in aclostridial toxin substrate useful in the invention. The donorfluorophore and acceptor generally are positioned to minimizeinterference with substrate binding to, or proteolysis by, theclostridial toxin. Thus, a donor fluorophore and acceptor can beselected and positioned, for example, so as to minimize the disruptionof bonded and non-bonded interactions that are important for binding,and to minimize steric hindrance. In addition, the spatial distancebetween the acceptor and donor fluorophore generally is limited toachieve efficient energy transfer from the donor fluorophore to theacceptor.

In standard nomenclature, the sequence surrounding a clostridial toxincleavage site is denoted P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′, with P₁-P₁′representing the scissile bond. In particular embodiments, the inventionprovides a substrate composition, cell or method that includes aclostridial toxin substrate in which the residue at position P₁, P₂, P₃,P₄, P₅, or P_(>5) is substitute with an amino acid conjugated to a donorfluorophore or acceptor, and in which the residue at position P₁′, P₂′,P₃′, P₄′, P₅′ or P_(>5)′ is substituted with an amino acid conjugated toa donor fluorophore or acceptor. In other embodiments, the inventionprovides a substrate composition, cell or method that incorporates aclostridial toxin substrate in which the residue at position P₁, P₃, P₄or P_(>5) is substituted with an amino acid conjugated to a donorfluorophore or acceptor, and in which the residue at position P₂′, P₃′,P₅′ or P_(>5)′ is substituted with an amino acid conjugated to a donorfluorophore or acceptor. It is further understood that the amino acidside chain of the residue conjugated to a donor fluorophore or acceptorcan be otherwise identical to the residue present in the correspondingposition of the naturally occurring target protein, or can contain, forexample, a different side chain. Further provided by the invention is asubstrate composition, cell or method that incorporates a clostridialtoxin substrate in which the residue at P₃, P₄ or P_(>5) is substitutedwith an amino acid conjugated to a donor fluorophore or acceptor, and inwhich the residue at position P₂′, P₃′, P₅′ or P_(>5)′ is substitutedwith an amino acid conjugated to a donor fluorophore or acceptor. Again,the amino acid side chain of the residue conjugated to the donorfluorophore or acceptor can be otherwise identical to the residuepresent in the corresponding position of the naturally occurring targetprotein, or can contain, for example, a different side chain.

As discussed above, efficiency of energy transfer from donor fluorophoreto acceptor is dependent, in part, on the spatial separation of thedonor fluorophore and acceptor molecules. As the distance between thedonor fluorophore and acceptor increases, there is less energy transferto the acceptor, and the donor fluorescence signal therefore increases,even prior to cleavage. The overall increase in fluorescence yield ofthe donor fluorophore, upon cleavage of the substrate, is dependent uponmany factors, including the separation distance between the donorfluorophore and acceptor in the substrate, the spectral overlap betweendonor fluorophore and acceptor, and the substrate concentration. Oneskilled in the art understands that, as the concentration of substrateincreases, intermolecular quenching of the donor, even after proteolyticcleavage, can become a factor. This phenomenon is denoted the “innerfilter effect.” One skilled in the art further understands that theintracellular concentration of substrate can be controlled, for example,through use of an inducible promoter or the external concentration ofsubstrate to which a cell is exposed.

The Förster distance, which is the separation between a donorfluorophore and an acceptor for 50% energy transfer, represents aspatial separation between donor fluorophore and acceptor that providesa good sensitivity. For peptide substrates, adjacent residues areseparated by a distance of approximately 3.6 A in the most extendedconformation. For example, the calculated Förster distance for afluorescein/tetramethylrhodamine pair is 55 A, which would represent aspatial separation between fluorescein and tetramethylrhodamine of about15 residues in the most extended conformation. Because peptides andpeptidomimetics in solution rarely have a fully extended conformation,donor fluorophores and acceptors can be more widely separated thanexpected based on a calculation performed using 3.6 A per residue andstill remain within the Förster distance as shown, for example, by theoccurrence of FRET between donor-acceptor pairs separated by about 50amino acids (Graham et al., Analyt. Biochem. 296: 208-217 (2001)).

Förster theory is based on very weak interactions between a donorfluorophore and an acceptor; spectroscopic properties such as absorptionof one fluorophore should not be altered in the presence of the other,defining the shortest distance range over which the theory is valid. Itis understood that, for many donor fluorophore-acceptor pairs, Förstertheory is valid when donor fluorophores and acceptors are separated byabout 10 A to 100 A. However, for particular donor fluorophore-acceptorpairs, Förster theory is valid below 10 A as determined by subpicosecondtechniques (Kaschke and Ernsting, Ultrafast Phenomenon in Spectroscopy(Klose and Wilhelmi (Eds.)) Springer-Verlag, Berlin 1990.

Thus, in particular embodiments, the invention provides a substratecomposition, cell or method that incorporates a clostridial toxinsubstrate in which the donor fluorophore is separated from the acceptorby a distance of at most 100 A. In other embodiments, the inventionprovides a substrate composition, cell or method that incorporates aclostridial toxin substrate in which the donor fluorophore is separatedfrom the acceptor by a distance of at most 90 A, 80 A, 70 A, 60 A, 50 A,40 A, 30 A or 20 A. In further embodiments, the invention provides asubstrate composition, cell or method that incorporates a clostridialtoxin substrate in which the donor fluorophore is separated from theacceptor by a distance of 10 A to 100 A, 10 A to 80 A, 10 A to 60 A, 10A to 40 A, 10 A to 20 A, 20 A to 100 A, 20 A to 80 A, 20 A to 60 A, 20 Ato 40 A, 40 A to 100 A, 40 A to 80 A or 40 A to 60 A. In still furtherembodiments, the invention provides a substrate composition, cell ormethod that incorporates a clostridial toxin substrate in which thedonor fluorophore and the acceptor are separated by at most sixresidues, at most eight residues, at most ten residues, at most twelveresidues, at most fifteen residues, at most twenty residues, at mosttwenty-five residues, at most thirty residues, at most thirty-fiveresidues, at most forty residues, at most forty-five residues, at mostfifty residues, at most sixty residues, at most seventy residues, atmost eighty residues, at most ninety residues, at most 100 residues, atmost 150 residues, at most 200 residues or up to the full-length of anaturally occurring clostridial toxin target protein.

One skilled in the art understands that a clostridial toxin substrateuseful in the invention can be designed to optimize the efficiency ofFRET as well as the ability to detect protease activity. One skilled inthe art understands that a donor fluorophore can be selected, ifdesired, with a high quantum yield, and acceptor can be selected, ifdesired, with a high extinction coefficient to maximize the Försterdistance. One skilled in the art further understands that fluorescencearising from direct excitation of an acceptor can be difficult todistinguish from fluorescence resulting from resonance energy transfer.Thus, it is recognized that a donor fluorophore and acceptor can beselected which have relatively little overlap of their excitationspectra such that the donor can be excited at a wavelength that does notresult in direct excitation of the acceptor. It further is recognizedthat a clostridial toxin substrate useful in the invention can bedesigned so that the emission spectra of the donor fluorophore andacceptor overlap relatively little such that the two emissions can bereadily distinguished. If desired, an acceptor having a highfluorescence quantum yield can be selected; such an acceptor can be usedadvantageously where acceptor fluorescence emission is to be detected asthe sole indicator of clostridial toxin activity, or as part of anemission ratio as discussed above.

It is understood that the donor fluorophore, acceptor, or both, can belocated within the active site cavity of botulinum or tetanus toxinholoenzyme. One skilled in the art understands that, if desired, aclostridial toxin substrate useful in the invention can be designed suchthat, when bound by toxin, the donor fluorophore, acceptor, or both, isexcluded from the active site cavity of toxin holoenzyme. As an example,a clostridial toxin substrate can include a botulinum toxin substrate ortetanus toxin substrate in which, when bound by toxin, the donorfluorophore, acceptor, or both, is excluded from the active site cavityof the toxin holoenzyme. The invention provides, for example, asubstrate composition, cell or method that incorporates a BoNT/A,BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G or TeNT substrate inwhich, when bound by toxin, the donor fluorophore, acceptor, or both, isexcluded from the active site cavity of toxin holoenzyme. In oneembodiment, the BoNT/A substrate contains at least six residues of humanSNAP-25, where the six residues include Gln₁₉₇-Arg₁₉₈, and furthercontains a donor fluorophore, acceptor, or both, which is positionedoutside of residues Arg₁₉₁ to Met₂₀₂, which can be within the activesite cavity of BoNT/A holoenzyme. In another embodiment, a BoNT/Bsubstrate contains at least six residues of VAMP-2, where the sixresidues include Gln₇₆-Phe₇₇, and further contains a donor fluorophore,acceptor, or both, which is positioned outside of residues Leu₇₀ toAla₈₁ of VAMP-2, which can be within the active site cavity of BoNT/Bholoenzyme.

In a complex of a VAMP substrate and the light chain of BoNT/B (LC/B),nearly all VAMP residues with side chains containing hydrogen bondacceptors or donors were hydrogen bonded with the LC/B. Thus, it isunderstood that a clostridial toxin substrate useful in the inventioncan be prepared, if desired, in which the potential for hydrogenbonding, for example, by Ser, Thr, Tyr, Asp, Glu, Asn or Gln residues isnot diminished in the clostridial toxin substrate as compared to anative protein sensitive to cleavage by the toxin. In particularembodiments, the present invention provides a substrate composition,cell or method incorporating a clostridial toxin substrate in which thepotential for hydrogen-bonding is not diminished in the substrate ascompared to a native protein sensitive to cleavage by the correspondingclostridial toxin.

It is understood that, in addition to a donor fluorophore, acceptor anda clostridial toxin recognition sequence, a clostridial toxin substrateuseful in the invention optionally can include one or more additionalcomponents. As an example, a flexible spacer sequence such as GGGGS (SEQID NO: 40) can be included in a clostridial toxin substrate useful inthe invention. A useful clostridial toxin substrate further can include,without limitation, one or more of the following: an affinity tag suchas HIS6, biotin, or an epitope such as FLAG, hemagluttinin (HA), c-myc,or AU1; an immunoglobulin hinge region; an N-hydroxysuccinimide linker;a peptide or peptidomimetic hairpin turn; or a hydrophilic sequence, oranother component or sequence that promotes the solubility or stabilityof the clostridial toxin substrate.

Methods for modifying proteins, peptides and peptidomimetics to containa donor fluorophore or acceptor are well known in the art (Faircloughand Cantor, Methods Enzymol. 48:347-379 (1978); Glaser et al., ChemicalModification of Proteins Elsevier Biochemical Press, Amsterdam (1975);Haugland, Excited States of Biopolymers (Steiner Ed.) pp. 29-58, PlenumPress, New York (1983); Means and Feeney, Bioconjugate Chem. 1:2-12(1990); Matthews et al., Methods Enzymol. 208:468-496 (1991); Lundblad,Chemical Reagents for Protein Modification 2nd Ed., CRC Press, BocaRatan, Fla. (1991); Haugland, supra, 1996). A variety of groups can beused to couple a donor fluorophore or acceptor, for example, to apeptide or peptidomimetic containing a clostridial toxin recognitionsequence. A thiol group, for example, can be used to couple a donorfluorophore or acceptor to the desired position in a peptide orpeptidomimetic to produce a clostridial toxin substrate useful in thesubstrate compositions, cells and methods of the invention. Haloacetyland maleimide labeling reagents also can be used to couple donorfluorophores or acceptors in preparing a clostridial toxin substrateuseful in the invention (see, for example, Wu and Brand, supra, 1994).

Donor fluorophores and acceptors including proteins such as GFP andallophycocyanin (APC) can be attached to a clostridial toxin recognitionsequence by a variety of means. A donor fluorophore or acceptor can beattached by chemical means, for example, using a cross-linker moiety.Cross-linkers are well known in the art, including homo- andhetero-bifunctional cross-linkers such as BMH and SPDP. One skilled inthe art understands that contaminating substrates containing only thedonor fluorophore can result in high fluorescence background. Suchbackground can be reduced or prevented, for example, by using a relativeexcess of acceptor to donor fluorophore in preparation of theclostridial toxin substrate. Where the donor fluorophore or acceptor isa protein, well known chemical methods for specifically linkingmolecules to the amino- or carboxy-terminus of a protein can beemployed. See, for example, “Chemical Approaches to Protein Engineering”in Protein Engineering: A Practical Approach Rees et al. (Eds) OxfordUniversity Press, 1992.

It is well known in the art that clostridial toxins have specific anddistinct cleavage sites. BoNT/A cleaves a Gln-Arg bond; BoNT/B and TeNTcleave a Gln-Phe bond; BoNT/C1 cleaves a Lys-Ala or Arg-Ala bond; BoNT/Dcleaves a Lys-Leu bond; BoNT/E cleaves an Arg-Ile bond; BoNT/F cleaves aGln-Lys bond; and BoNT/G cleaves an Ala-Ala bond (see Table D). Instandard nomenclature, the sequence surrounding a clostridial toxincleavage site is denoted P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′, with P₁-P₁′representing the scissile bond. It is understood that a P₁ or P₁′ site,or both, can be substituted with another amino acid or amino acidmimetic in place of the naturally occurring residue. As an example,BoNT/A substrates have been prepared in which the P₁ position (Gln) ismodified to be an alanine, 2-aminobutyric acid or asparagine residue;these substrates were hydrolyzed by BoNT/A at the P₁-Arg bond (Schmidtand Bostian, J. Protein Chem. 16:19-26 (1997)). While it is recognizedthat substitutions can be introduced at the P₁ position of the scissilebond, for example, a BoNT/A scissile bond, it is further recognized thatconservation of the P₁′ residue can be advantageous (Vaidyanathan etal., J. Neurochem. 72:327-337 (1999)). Thus, in particular embodiments,the invention provides a substrate composition, cell or method whichrelies on a clostridial toxin substrate having a clostridial toxinrecognition sequence in which the P₁′ residue is not modified orsubstituted relative to the naturally occurring residue in a targetprotein cleaved by the clostridial toxin. In other embodiments, theinvention provides a substrate composition, cell or method which relieson a clostridial toxin substrate having a recognition sequence in whichthe P₁ residue is modified or substituted relative to the naturallyoccurring residue in a target protein cleaved by the clostridial toxin;such a clostridial toxin substrate retains susceptibility to peptidebond cleavage between the P₁ and P₁′ residues. TABLE D Bond cleaved inhuman VAMP-2, SNAP-25 or Syntaxin Toxin Target P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′ SEQ ID NO BoNT/A SNAP-25Glu-Ala-Asn-Gln-Arg*-Ala-Thr-Lys SEQ ID NO: 41 BoNT/B VAMP-2Gly-Ala-Ser-Gln-Phe*-Glu-Thr-Ser SEQ ID NO: 42 BoNT/C1 syntaxinAsp-Thr-Lys-Lys-Ala*-Val-Lys-Tyr SEQ ID NO: 43 BoNT/D VAMP-2Arg-Asp-Gln-Lys-Leu*-Ser-Glu-Leu SEQ ID NO: 44 BoNT/E SNAP-25Gln-Ile-Asp-Arg-Ile*-Met-Glu-Lys SEQ ID NO: 45 BoNT/F VAMP-2Glu-Arg-Asp-Gln-Lys*-Leu-Ser-Glu SEQ ID NO: 46 BoNT/G VAMP-2Glu-Thr-Ser-Ala-Ala*-Lys-Leu-Lys SEQ ID NO: 47 TeNT VAMP-2Gly-Ala-Ser-Gln-Phe*-GLu-Thr-Ser SEQ ID NO: 48* Scissile bond shown in bold

SNAP-25, VAMP and syntaxin share a short motif located within regionspredicted to adopt an α-helical conformation (see FIG. 4). This motif ispresent in SNAP-25, VAMP and syntaxin isoforms expressed in animalssensitive to the neurotoxins. In contrast, Drosophila and yeast homologsthat are resistant to these neurotoxins and syntaxin isoforms notinvolved in exocytosis contain sequence variations in the α-helicalmotif regions of these VAMP and syntaxin proteins.

Multiple repetitions of the α-helical motif are present in proteinssensitive to cleavage by clostridial toxins: Four copies are naturallypresent in SNAP-25; two copies are naturally present in VAMP; and twocopies are naturally present in syntaxin (see FIG. 4A). Furthermore,peptides corresponding to the specific sequence of the α-helical motifscan inhibit neurotoxin activity in vitro and in vivo, and such peptidescan cross-inhibit different neurotoxins. In addition, antibodies raisedagainst such peptides can cross-react among the three target proteins,indicating that this α-helical motif is exposed on the cell surface andadopts a similar configuration in each of the three target proteins.Consistent with these findings, SNAP-25-specific, VAMP-specific andsyntaxin-specific neurotoxins cross-inhibit each other by competing forthe same binding site, although they do not cleave targetsnon-specifically. These results indicate that a clostridial toxinrecognition sequence can include, if desired, at least one α-helicalmotif. It is recognized that an α-helical motif is not required forcleavage by a clostridial toxin, as evidenced by 16-mer and 17-mersubstrates for BoNT/A, discussed further below.

Although multiple α-helical motifs are found in the naturally occurringSNAP-25, VAMP and syntaxin target proteins, a clostridial toxinrecognition sequence useful in the substrate compositions, cells andmethods of the invention can have a single α-helical motif. Inparticular embodiments, the invention relies on a clostridial toxinrecognition sequence including two or more α-helical motifs. A BoNT/A orBoNT/E recognition sequence can include, for example, the S4 α-helicalmotif, alone or combined with one or more additional α-helical motifs; aBoNT/B, BoNT/G or TeNT recognition sequence can include, for example,the V2 α-helical motif, alone or combined with one or more additionalα-helical motifs; a BoNT/C1 recognition sequence can include, forexample, the S4 α-helical motif, alone or combined with one or moreadditional α-helical motifs, or X2 α-helical motif, alone or combinedwith one or more additional α-helical motifs; and a BoNT/D or BoNT/Frecognition sequence can include, for example, the V1 α-helical motif,alone or combined with one or more additional α-helical motifs (see FIG.4A).

A variety of BoNT/A substrates are useful in the invention. A BoNT/Asubstrate useful in the substrate compositions, cells and methods of theinvention can include, for example, at least six consecutive residues ofSNAP-25, where the six consecutive residues include Gln-Arg, or apeptidomimetic thereof. Such a BoNT/A substrate can have, for example,at least six consecutive residues of human SNAP-25, where the sixconsecutive residues include Gln₁₉₇-Arg₁₉₈, or a peptidomimetic thereof.Such a BoNT/A substrate can include, for example, the amino acidsequence Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys (SEQ ID NO: 41) or, forexample, residues 187 to 203 of human SNAP-25 (SEQ ID NO: 4). A BoNT/Asubstrate further can include, if desired, a carboxy-terminal amide.

As used herein, the term “botulinum toxin serotype A recognitionsequence” is synonymous with “BoNT/A recognition sequence” and means ascissile bond together with adjacent or non-adjacent recognitionelements sufficient for detectable proteolysis at the scissile bond by aBoNT/A under conditions suitable for clostridial toxin proteaseactivity. A scissile bond cleaved by BoNT/A can be, for example,Gln-Ala.

A variety of BoNT/A recognition sequences are well known in the art andare useful in the invention. A BoNT/A recognition sequence can have, forexample, residues 134 to 206 or residues 137 to 206 of human SNAP-25(Ekong et al., supra, 1997; U.S. Pat. No.5,962,637). A BoNT/Arecognition sequence also can include, without limitation, the sequenceThr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met (SEQ ID NO: 49) or apeptidomimetic thereof, which corresponds to residues 190 to 202 ofhuman SNAP-25;Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys (SEQ ID NO:50) or a peptidomimetic thereof, which corresponds to residues 187 to201 of human SNAP-25;Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met (SEQ IDNO: 51) or a peptidomimetic thereof, which corresponds to residues 187to 202 of human SNAP-25;Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu (SEQID NO: 52) or a peptidomimetic thereof, which corresponds to residues187 to 203 of human SNAP-25;Asp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met (SEQID NO: 53) or a peptidomimetic thereof, which corresponds to residues186 to 202 of human SNAP-25; orAsp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu(SEQ ID NO: 54) or a peptidomimetic thereof, which corresponds toresidues 186 to 203 of human SNAP-25. See, for example, Schmidt andBostian, J. Protein Chem. 14:703-708 (1995); Schmidt and Bostian, supra,1997; Schmidt et al., FEBS Letters 435:61-64 (1998); and Schmidt andBostian, U.S. Pat. No. 5,965,699). If desired, a similar BoNT/Arecognition sequence can be prepared from a corresponding (homologous)segment of another BoNT/A-sensitive SNAP-25 isoform or homolog such as,for example, murine, rat, goldfish or zebrafish SNAP-25 or can be any ofthe peptides described herein or known in the art, for example, in U.S.Pat. No. 5,965,699. TABLE E Cleavage of SNAP-25 and relatedproteins^(a, b, c, d) Resistance to Species ————— Isoform Cleavage SitesSEQ ID NO: Cleavage by       BoNT/E           BoNT/A  BoNT/C            

                

human mouse ——————— SNAP-25 ¹⁷⁴qnrqid ri mekadsnktridean qra tkmlgsg²⁰⁶none^(a) rat human ——————— SNAP-23 ¹⁸⁰qnpqik ri tdkadtnrdridian arakklids^(end) all^(b) mouse ——————— SNAP-23 ¹⁷⁹qnqqiq ki tekadtnknridiantra kklids^(end) BoNT/A & C chicken ————— SNAP-25 ¹⁷⁴qnrqid rimeklipikpglmkpt svq qrcsavvk^(end) BoNT/A & C goldfish ———— SNAP-25A¹⁷¹qnrqid ri mdmadsnktridean qra tkmlgsg^(end) none goldfish———— SNAP-25B ¹⁷²qnrqid ri mekadsnktridean qra tkmlgsg^(end) noneTorpedo ————— SNAP-25 ¹⁸⁰qnaqvd ri vvkgdmnkaridean kha tkml^(end)BoNT/E^(c) & A sea urchin —— SNAP-25 ¹⁸⁰qnsqvg ri tskaesnegrinsad kraknilrnk^(end) (?)^(e) C-elegans ——— SNAP-25 ²⁰³qnrqld ri hdkqsnevrvesankrak nlitk^(end) BoNT/A & C Drosophila —— SNAP-25 ¹⁸²qnrqid rinrkgesneariavan qra hqllk^(end) BoNT/E & A^(e) leech ——————— SNAP-25¹⁸¹qnrqvd ri nnkmtsnqlrisdan kra skllke^(end) BoNT/A^(e)^(a) = In vitro cleavage of SNAP-25 requires 1000-fold higher BoNT/Cconcentration than BoNT/A or /E.^(b) = Substitution of p182r, or k185dd (boxes) induces susceptibilitytoward BoNT/E.^(c) = Resistance to BoNT/A possibly due to d189 or e189 substitution byv189, see box.^(d) = Note that Torpedo is susceptible to BoNT/A.^(e) = Note the presence of several non-conservative mutations aroundputative cleavage sites.

A BoNT/A recognition sequence useful in the invention can correspond toa segment of a protein that is sensitive to cleavage by botulinum toxinserotype A, or can be substantially similar to a segment of aBoNT/A-sensitive protein. As illustrated in Table E, a variety ofnaturally occurring proteins sensitive to cleavage by BoNT/A are knownin the art and include, for example, human, mouse and rat SNAP-25; andgoldfish SNAP-25A and SNAP-25B. Thus, a BoNT/A recognition sequenceuseful in the invention can correspond, for example, to a segment ofhuman SNAP-25, mouse SNAP-25, rat SNAP-25, goldfish SNAP-25A or 25B, oranother naturally occurring protein sensitive to cleavage by BoNT/A.Furthermore, comparison of native SNAP-25 amino acid sequences cleavedby BoNT/A reveals that such sequences are not absolutely conserved (seeTable E and FIG. 5), indicating that a variety of amino acidsubstitutions and modifications relative to a naturally occurringBoNT/A-sensitive SNAP-25 sequence can be tolerated in a BoNT/Arecognition sequence useful in the invention.

A clostridial toxin substrate, such as a BoNT/A substrate, can have oneor multiple modifications as compared to a naturally occurring sequencethat is cleaved by the corresponding clostridial toxin. As an example,as compared to a 17-mer corresponding to residues 187 to 203 of humanSNAP-25, substitution of Asp193 with Asn in the BoNT/A substrateresulted in a relative rate of proteolysis of 0.23; substitution ofGlu194 with Gln resulted in a relative rate of 2.08; substitution ofAla195 with 2-aminobutyric acid resulted in a relative rate of 0.38; andsubstitution of Gln197 with Asn, 2-aminobutyric acid or Ala resulted ina relative rate of 0.66, 0.25, or 0.19, respectively (see Table F).Furthermore, substitution of Ala199 with 2-aminobutyric acid resulted ina relative rate of 0.79; substitution of Thr200 with Ser or2-aminobutyric acid resulted in a relative rate of 0.26 or 1.20,respectively; substitution of Lys201 with Ala resulted in a relativerate of 0.12; and substitution of Met202 with Ala or norleucine resultedin a relative rate of 0.38 or 1.20, respectively. See Schmidt andBostian, supra, 1997. These results indicate that a variety of residuescan be substituted in a clostridial toxin substrate as compared to anaturally occurring toxin-sensitive sequence. In the case of BoNT/A,these results indicate that residues including but not limited toGlu194, Ala195, Gln197, Ala199, Thr200 and Met202, Leu203, Gly204,Ser205, and Gly206, as well as residues more distal from the Gln-Argscissile bond, can be substituted or conjugated to a donor fluorophoreor acceptor in a BoNT/A substrate useful in the invention. Such a BoNT/Asubstrate is detectably proteolyzed at the scissile bond by BoNT/A underconditions suitable for clostridial toxin protease activity. Thus, aBoNT/A substrate can include, if desired, one or several amino acidsubstitutions, additions or deletions relative to a naturally occurringSNAP-25 sequence. TABLE F Kinetic parameters of BoNT/A synthetic peptidesubstrates Relative Peptide Sequence^(a) SEQ ID NO: Rate^(b) [1-15]SNKTRIDEANQRATK 28 0.03 [1-16] SNKTRIDEANQRATKM 29 1.17 [1-17]SNKTRIDEANQRATKML 30 1.00 M16A SNKTRIDEANQRATK A L 44 0.38 M16XSNKTRIDEANQRATK X L 45 1.20 K15A SNKTRIDEANQRAT A ML 46 0.12 T14SSNKTRIDEANQRA S KML 47 0.26 T14B SNKTRIDEANQRA B KML 48 1.20 A13BSNKTRIDEANQR B TKML 49 0.79 Q11A SNKTRIDEAN A RATKML 50 0.19 Q11BSNKTRIDEAN B RATKML 51 0.25 Q11N SNKTRIDEAN N RATKML 52 0.66 N10ASNKTRIDEA A QRATKML 53 0.06 A9B SNKTRIDE B NQRATKML 54 0.38 E8Q SNKTRIDQ ANQRATKML 55 2.08 D7N SNKTRI N EANQRATKML 56 0.23^(a)Nonstandard amino acid abbreviations are: B 2-aminobutyric acid;X 2-aminohexanoic acid (norleucine)^(b)Initial hydrolysis rates relative to peptide [1-17]. Peptideconcentrations were 1.0 mM.

A variety of BoNT/B substrates are useful in the invention. A BoNT/Bsubstrate useful in the invention can have, for example, at least sixconsecutive residues of VAMP, where the six consecutive residues includeGln-Phe, or a peptidomimetic thereof. As an example, a BoNT/B substratecan contain at least six consecutive residues of human VAMP-2, the sixconsecutive residues including Gln₇₆-Phe₇₇, or a peptidomimetic thereof.In one embodiment, a BoNT/B substrate includes the amino acid sequenceGly-Ala-Ser-Gln-Phe-Glu-Thr-Ser (SEQ ID NO: 42), or a peptidomimeticthereof. In other embodiments, a BoNT/B substrate includes residues 55to 94 of human VAMP-2 (SEQ ID NO: 11); residues 60 to 94 of human VAMP-2(SEQ ID NO: 11); or residues 60 to 88 of human VAMP-2 (SEQ ID NO: 11),or a peptidomimetic of one of these sequences.

As used herein, the term “botulinum toxin serotype B recognitionsequence” is synonymous with “BoNT/B recognition sequence” and means ascissile bond together with adjacent or non-adjacent recognitionelements sufficient for detectable proteolysis at the scissile bond by aBoNT/B under appropriate conditions. A scissile bond cleaved by BoNT/Bcan be, for example, Gln-Phe.

A variety of BoNT/B recognition sequences are well known in the art orcan be defined by routine methods. Such a BoNT/B recognition sequencecan include, for example, a sequence corresponding to some or all of thehydrophilic core of a VAMP protein such as human VAMP-1 or human VAMP-2.A BoNT/B recognition sequence can include, without limitation, residues33 to 94, residues 45 to 94, residues 55 to 94, residues 60 to 94,residues 65 to 94, residues 60 to 88 or residues 65 to 88 of humanVAMP-2 (SEQ ID NO: 11), or residues 60 to 94 of human VAMP-1 (SEQ ID NO:10). See, for example, Shone et al., Eur. J. Biochem. 217: 965-971(1993). and U.S. Pat. No. 5,962,637. If desired, a similar BoNT/Brecognition sequence can be prepared from a corresponding (homologous)segment of another BoNT/B-sensitive VAMP isoform or homolog such ashuman VAMP-1 or rat or chicken VAMP-2.

Thus, it is understood that a BoNT/B recognition sequence can correspondto a segment of a protein that is sensitive to cleavage by botulinumtoxin serotype B, or can be substantially similar to such a segment of aBoNT/B-sensitive protein. As shown in Table G, a variety of naturallyoccurring proteins sensitive to cleavage by BoNT/B are known in the artand include, for example, human, mouse and bovine VAMP-1 and VAMP-2; ratVAMP-2; rat cellubrevin; chicken VAMP-2; Torpedo VAMP-1; sea urchinVAMP; Aplysia VAMP; squid VAMP; C. elegans VAMP; Drosophila n-syb; andleech VAMP. Thus, a BoNT/B recognition sequence included in a BoNT/Bsubstrate can correspond, for example, to a segment of human VAMP-1 orVAMP-2, mouse VAMP-1 or VAMP-2, bovine VAMP-1 or VAMP-2, rat VAMP-2, ratcellubrevin, chicken VAMP-2, Torpedo VAMP-1, sea urchin VAMP, AplysiaVAMP, squid VAMP, C. elegans VAMP, Drosophila n-syb, leech VAMP, oranother naturally occurring protein sensitive to cleavage by BoNT/B.Furthermore, as shown in Table G, comparison of native VAMP amino acidsequences cleaved by BoNT/B reveals that such sequences are notabsolutely conserved (see, also, FIG. 6), indicating that a variety ofamino acid substitutions and modifications relative to a naturallyoccurring VAMP sequence can be tolerated in a BoNT/B substrate useful,for example, in a BoNT/B substrate composition of the invention. TABLE GCleavage of VAMP^(a, b) Resistance to Species —————— Isoform CleavageSites SEQ ID NO: Cleavage by                          BoNT/B    BoNT/F  BoNT/D         TeNT       BoNT/G           

                   

     

human mouse ——————— VAMP-1 ⁵³dkvlerd qkl selddradalqagas qf ess aaklkrkyww⁹² none bovine human mouse ——————— VAMP-2 ⁵¹dkvlerd qklselddradalqagas qf ets aa klkrkyww⁹⁰ none bovine rat ————————— VAMP-2⁵³dkvlerd qkl selddradalqagas vf ess aa klkrkyww⁹² TeNT & BONT/B rat————————— VAMP-2 ⁵¹dkvlerd qkl selddradalqagas qf ets aa klkrkyww⁹⁰ nonerat ———— Cellubrevin ³⁸dkvlerd qkl selddradalqagas qf ets aa klkrkyww⁷⁷none rat ———————— TI-VAMP ¹⁴⁶dlvaqrg eel ellidktenlvdssv tf ktt srnlaramcm¹⁷⁵ all chicken ————— VAMP-1 ^(—)----erd qkl selddradalqagas vfess aa klkr----^(—) TeNT & BoNT/B chicken ————— VAMP-2 ^(—)----erd qklselddradalqagas qf ets aa klkr----^(—) none Torpedo ————— VAMP-1⁵⁵dkvlerd qkl selddradalqagas qf ess aa klkrkyww⁹⁴ none sea urchin———— VAMP ³⁵dkvldrd qal svlddradalqqgas qf etn ag klkrkyww⁷⁴ BoNT/F, D &G Aplysia ——————— VAMP ⁴¹ekvldrd qki sqlddraealqagas qf eas ag klkrkywwBoNT/G squid ————————— VAMP ⁸⁰dkvlerd ski selddradalqagas qf eas agklkrkfww⁹⁹ BoNT/F & G C. elegans ———— VAMP ⁸⁶nkvmerd vql nsldhraevlqngasqf qqs sr elkrqyww²¹⁵ BoNT/F, D & G Drosphila ————— syb^(a) ⁶⁷ekvlerdqkl selgeradqleqgas qs eqq ag klkrkqww¹⁰⁶ TeNT & BoNT/B & G Drosphila——— n-syb^(b) ⁶¹ekvlerd skl selddradalqqgas qf eqq ag klkrkfwl¹⁰⁰ BoNT/F& G leech ————————— VAMP ⁴⁹dkvlekd qkl aeldgradalqagas qf eas agklkrkfww⁸⁸ BoNT/G^(a) = Sequence corrected in position 93 (f > s).^(b) = Sequence corrected in position 68 (t > s).

Various BoNT/C1 substrates are useful in the invention. A BoNT/C1substrate useful in the invention can have, for example, at least sixconsecutive residues of syntaxin, the six consecutive residues includingLys-Ala, or a peptidomimetic thereof. As an example, a BoNT/C1 substratecan have at least six consecutive residues of human syntaxin, the sixconsecutive residues including Lys₂₅₃-Ala₂₅₄, or a peptidomimeticthereof. In one embodiment, a BoNT/C1 substrate contains the amino acidsequence Asp-Thr-Lys-Lys-Ala-Val-Lys-Tyr (SEQ ID NO: 43), or apeptidomimetic thereof.

A BoNT/C1 substrate also can contain, for example, at least sixconsecutive residues of SNAP-25, where the six consecutive residuesinclude Arg-Ala, or a peptidomimetic thereof. Such a BoNT/C1 substratecan have, for example, at least six consecutive residues of humanSNAP-25, the six consecutive residues including Arg₁₉₈-Ala₁₉₉, or apeptidomimetic thereof. In one embodiment, a BoNT/C1 substrate containsresidues 93 to 202 of human SNAP-25 (SEQ ID NO: 4), or a peptidomimeticthereof.

As used herein, the term “botulinum toxin serotype C1 recognitionsequence” is synonymous with “BoNT/C1 recognition sequence” and means ascissile bond together with adjacent or non-adjacent recognitionelements sufficient for detectable proteolysis at the scissile bond by aBoNT/C1 under appropriate conditions. A scissile bond cleaved by BoNT/C1can be, for example, Lys-Ala or Arg-Ala.

It is understood that a BoNT/C1 recognition sequence can correspond to asegment of a protein that is sensitive to cleavage by botulinum toxinserotype C1, or can be substantially similar to a segment of aBoNT/C1-sensitive protein. As shown in Table H, a variety of naturallyoccurring proteins sensitive to cleavage by BoNT/C1 are known in the artand include, for example, human, rat, mouse and bovine syntaxin 1A and1B; rat syntaxins 2 and 3; sea urchin syntaxin; Aplysia syntaxin 1;squid syntaxin; Drosophila Dsynt1; and leech syntaxin 1. Thus, a BoNT/C1recognition sequence useful in a BoNT/C1 substrate can correspond, forexample, to a segment of human, rat, mouse or bovine syntaxin 1A or 1B,rat syntaxin 2, rat syntaxin 3, sea urchin syntaxin, Aplysia syntaxin 1,squid syntaxin, Drosophila Dsynt1, leech syntaxin 1, or anothernaturally occurring protein sensitive to cleavage by BoNT/C1.Furthermore, comparison of native syntaxin amino acid sequences cleavedby BoNT/C1 reveals that such sequences are not absolutely conserved (seeTable H and FIG. 7), indicating that a variety of amino acidsubstitutions and modifications relative to a naturally occurringBoNT/C1-sensitive syntaxin sequence can be tolerated in a BoNT/C1substrate useful in the invention.

A variety of naturally occurring SNAP-25 proteins also are sensitive tocleavage by BoNT/C1, including human, mouse and rat SNAP-25; goldfishSNAP-25A and 25B; and Drosophila and leech SNAP-25. Thus, a BoNT/C1recognition sequence useful in a BoNT/C1 substrate can correspond, forexample, to a segment of human, mouse or rat SNAP-25, goldfish SNAP-25Aor 25B, Torpedo SNAP-25, zebrafish SNAP-25, Drosophila SNAP-25, leechSNAP-25, or another naturally occurring protein sensitive to cleavage byBoNT/C1. As discussed above in regard to variants of naturally occurringsyntaxin sequences, comparison of native SNAP-25 amino acid sequencescleaved by BoNT/C1 reveals significant sequence variability (see Table Eand FIG. 5 above), indicating that a variety of amino acid substitutionsand modifications relative to a naturally occurring BoNT/C1-sensitiveSNAP-25 sequence can be tolerated in a BoNT/C1 substrate useful in theinvention.

One skilled in the art appreciates that a variety of BoNT/D substratesare useful in the invention. A BoNT/D substrate useful in the inventioncan have, for example, at least six consecutive residues of VAMP, thesix consecutive residues including Lys-Leu, or a peptidomimetic thereof.In one embodiment, a BoNT/D substrate contains at least six consecutiveresidues of human VAMP, the six consecutive residues includingLys₅₉-Leu₆₀, or a peptidomimetic thereof. In another embodiment, aBoNT/D substrate contains the amino acid sequenceArg-Asp-Gln-Lys-Leu-Ser-Glu-Leu (SEQ ID NO: 44), or a peptidomimeticthereof. In a further embodiment, a BoNT/D substrate includes residues27 to 116 of rat VAMP-2 (SEQ ID NO: 109), or a peptidomimetic thereof.

The term “botulinum toxin serotype D recognition sequence” is synonymouswith “BoNT/D recognition sequence” and means a scissile bond togetherwith adjacent or non-adjacent recognition elements sufficient fordetectable proteolysis at the scissile bond by a BoNT/D underappropriate conditions. A scissile bond cleaved by BoNT/D can be, forexample, Lys-Leu. TABLE H Cleavage of syntaxin Resistance to Species———————— Isoform Cleavage Sites SEQ ID NO: Cleavage by         BoNT/C              

human rat ———————— syntaxin 1A ²⁴⁵eravsdtk ka vkyqskar²⁶² no mousebovine human rat ———————— syntaxin 1B ²⁴⁴eravsdtk ka vkyqskar²⁶¹ nomouse bovine rat ————————— syntaxin 2 ²⁴⁵ehakeetk ka ikyqskar²⁶² no rat————————— syntaxin 3 ²⁴⁴ekardetr ka mkyqgqar²⁶¹ no rat————————— syntaxin 4 ²⁴⁴ergqehvk ia lenqkkar²⁶¹ yes chicken———— syntaxin 1B ²³⁹vpevfvtk sa vmyqcksr²⁵⁹ expected sea urchin———— syntaxin ²⁴³vrrqndtk ka vkyqskar²⁶⁰ no Aplysia ————— syntaxin 1²⁴⁷etakmdtk ka vkyqskar²⁶⁴ no squid ————————— syntaxin ²⁴⁸etakvdtk kavkyqskar²⁶⁵ no Drosophila ————— Dsynt 1 ²⁴⁸qtatqdtk ka lkyqskar²⁶⁵ noleech ——————— syntaxin 1 ²⁵¹etaaadtk ka mkyqsaar²⁶⁸ no

A variety of BoNT/D recognition sequences are well known in the art orcan be defined by routine methods. A BoNT/D recognition sequence caninclude, for example, residues 27 to 116; residues 37 to 116; residues 1to 86; residues 1 to 76; or residues 1 to 69 of rat VAMP-2 (SEQ ID NO:109; Yamasaki et al., J. Biol. Chem. 269:12764-12772 (1994)). Thus, aBoNT/D recognition sequence can include, for example, residues 27 to 69or residues 37 to 69 of rat VAMP-2 (SEQ ID NO: 109). If desired, asimilar BoNT/D recognition sequence can be prepared from a corresponding(homologous) segment of another BoNT/D-sensitive VAMP isoform or homologsuch as human VAMP-1 or human VAMP-2.

A BoNT/D recognition sequence can correspond to a segment of a proteinthat is sensitive to cleavage by botulinum toxin serotype D, or can besubstantially similar to a segment of a BoNT/D-sensitive protein. Asshown in Table H, a variety of naturally occurring proteins sensitive tocleavage by BoNT/D are known in the art and include, for example, human,mouse and bovine VAMP-1 and VAMP-2; rat VAMP-1 and VAMP-2; ratcellubrevin; chicken VAMP-1 and VAMP-2; Torpedo VAMP-1; Aplysia VAMP;squid VAMP; Drosophila syb and n-syb; and leech VAMP. Thus, a BoNT/Drecognition sequence can correspond, for example, to a segment of humanVAMP-1 or VAMP-2, mouse VAMP-1 or VAMP-2, bovine VAMP-1 or VAMP-2, ratVAMP-1 or VAMP-2, rat cellubrevin, chicken VAMP-1 or VAMP-2, TorpedoVAMP-1, Aplysia VAMP, squid VAMP, Drosophila syb or n-syb, leech VAMP,or another naturally occurring protein sensitive to cleavage by BoNT/D.Furthermore, as shown in Table H above, comparison of native VAMP aminoacid sequences cleaved by BoNT/D reveals significant sequencevariability (see, also, FIG. 6), indicating that a variety of amino acidsubstitutions and modifications relative to a naturally occurringBoNT/D-sensitive VAMP sequence can be tolerated in a BoNT/D substrateuseful in the invention.

A variety of BoNT/E substrates are useful in the invention. A BoNT/Esubstrate can contain, for example, at least six consecutive residues ofSNAP-25, the six consecutive residues including Arg-Ile, or apeptidomimetic thereof. Such a BoNT/E substrate can have, for example,at least six consecutive residues of human SNAP-25, the six consecutiveresidues including Arg₁₈₀-Ile₁₈₁, or a peptidomimetic thereof. Inparticular embodiments, a BoNT/E substrate includes the amino acidsequence Gln-Ile-Asp-Arg-Ile-Met-Glu-Lys (SEQ ID NO: 45), or apeptidomimetic thereof. In other embodiments, a BoNT/E substrateincludes residues 156 to 186 of human SNAP-25 (SEQ ID NO: 4), or apeptidomimetic thereof.

As used herein, the term “botulinum toxin serotype E recognitionsequence” is synonymous with “BoNT/E recognition sequence” and means ascissile bond together with adjacent or non-adjacent recognitionelements sufficient for detectable proteolysis at the scissile bond by aBoNT/E under appropriate conditions. A scissile bond cleaved by BoNT/Ecan be, for example, Arg-Ile.

One skilled in the art appreciates that a BoNT/E recognition sequencecan correspond to a segment of a protein that is sensitive to cleavageby botulinum toxin serotype E, or can be substantially similar to asegment of a BoNT/E-sensitive protein. A variety of naturally occurringproteins sensitive to cleavage by BoNT/E are known in the art andinclude, for example, human, mouse and rat SNAP-25; mouse SNAP-23;chicken SNAP-25; goldfish SNAP-25A and SNAP-25B; zebrafish SNAP-25; C.elegans SNAP-25; and leech SNAP-25 (see Table E). Thus, a BoNT/Erecognition sequence can correspond, for example, to a segment of humanSNAP-25, mouse SNAP-25, rat SNAP-25, mouse SNAP-23, chicken SNAP-25,goldfish SNAP-25A or 25B, C. elegans SNAP-25, leech SNAP-25, or anothernaturally occurring protein sensitive to cleavage by BoNT/E.Furthermore, as shown in Table E and FIG. 5 above, comparison of nativeSNAP-23 and SNAP-25 amino acid sequences cleaved by BoNT/E reveals thatsuch sequences are not absolutely conserved, indicating that a varietyof amino acid substitutions and modifications relative to a naturallyoccurring BoNT/E-sensitive SNAP-23 or SNAP-25 sequence can be toleratedin a BoNT/E substrate useful in the invention.

A variety of useful BoNT/F substrates can be useful in the invention.Such BoNT/F substrates can include, for example, at least sixconsecutive residues of VAMP, the six consecutive residues includingGln-Lys, or a peptidomimetic thereof. In one embodiment, a BoNT/Fsubstrate has at least six consecutive residues of human VAMP, the sixconsecutive residues including Gln₅₈-Lys₅₉, or a peptidomimetic thereof.In another embodiment, a BoNT/F substrate includes residues 27 to 116 ofrat VAMP-2 (SEQ ID NO: 109), or a peptidomimetic thereof. In a furtherembodiment, a BoNT/F substrate includes the amino acid sequenceGlu-Arg-Asp-Gln-Lys-Leu-Ser-Glu (SEQ ID NO: 46), or a peptidomimeticthereof.

The term “botulinum toxin serotype F recognition sequence,” as usedherein, is synonymous with “BoNT/F recognition sequence” and means ascissile bond together with adjacent or non-adjacent recognitionelements sufficient for detectable proteolysis at the scissile bond by aBoNT/F under appropriate conditions. A scissile bond cleaved by BoNT/Fcan be, for example, Gln-Lys.

A variety of BoNT/F recognition sequences are well known in the art orcan be defined by routine methods. A BoNT/F recognition sequence caninclude, for example, residues 27 to 116; residues 37 to 116; residues 1to 86; residues 1 to 76; or residues 1 to 69 of rat VAMP-2 (SEQ ID NO:109; Yamasaki et al., supra, 1994). A BoNT/F recognition sequence alsocan include, for example, residues 27 to 69 or residues 37 to 69 of ratVAMP-2 (SEQ ID NO: 109). It is understood that a similar BoNT/Frecognition sequence can be prepared, if desired, from a corresponding(homologous) segment of another BoNT/F-sensitive VAMP isoform or homologsuch as human VAMP-1 or human VAMP-2.

A BoNT/F recognition sequence can correspond to a segment of a proteinthat is sensitive to cleavage by botulinum toxin serotype F, or can besubstantially similar to a segment of a BoNT/F-sensitive protein. Avariety of naturally occurring proteins sensitive to cleavage by BoNT/Fare known in the art and include, for example, human, mouse and bovineVAMP-1 and VAMP-2; rat VAMP-1 and VAMP-2; rat cellubrevin; chickenVAMP-1 and VAMP-2; Torpedo VAMP-1; Aplysia VAMP; Drosophila syb; andleech VAMP (see Table H). Thus, a BoNT/F recognition sequence cancorrespond, for example, to a segment of human VAMP-1 or VAMP-2, mouseVAMP-1 or VAMP-2, bovine VAMP-1 or VAMP-2, rat VAMP-1 or VAMP-2, ratcellubrevin, chicken VAMP-1 or VAMP-2, Torpedo VAMP-1, Aplysia VAMP,Drosophila syb, leech VAMP, or another naturally occurring proteinsensitive to cleavage by BoNT/F. Furthermore, as shown in Table H above,comparison of native VAMP amino acid sequences cleaved by BoNT/F revealsthat such sequences are not absolutely conserved (see, also, FIG. 6),indicating that a variety of amino acid substitutions and modificationsrelative to a naturally occurring BoNT/F-sensitive VAMP sequence can betolerated in a BoNT/F substrate useful in the invention.

As for other clostridial toxin substrates, a variety of BoNT/Gsubstrates can be useful in the invention. A BoNT/G substrate useful inthe invention can have, for example, at least six consecutive residuesof VAMP, the six consecutive residues including Ala-Ala, or apeptidomimetic thereof. Such a BoNT/G substrate can have, for example,at least six consecutive residues of human VAMP, the six consecutiveresidues including Ala₈₃-Ala₈₄, or a peptidomimetic thereof. In oneembodiment, a BoNT/G substrate contains the amino acid sequenceGlu-Thr-Ser-Ala-Ala-Lys-Leu-Lys (SEQ ID NO: 47), or a peptidomimeticthereof.

As used herein, the term “botulinum toxin serotype G recognitionsequence” is synonymous with “BoNT/G recognition sequence” and means ascissile bond together with adjacent or non-adjacent recognitionelements sufficient for detectable proteolysis at the scissile bond by aBoNT/G under appropriate conditions. A scissile bond cleaved by BoNT/Gcan be, for example, Ala-Ala.

A BoNT/G recognition sequence can correspond to a segment of a proteinthat is sensitive to cleavage by botulinum toxin serotype G, or can besubstantially similar to such a BoNT/G-sensitive segment. Asillustration in Table H above, a variety of naturally occurring proteinssensitive to cleavage by BoNT/G are known in the art and include, forexample, human, mouse and bovine VAMP-1 and VAMP-2; rat VAMP-1 andVAMP-2; rat cellubrevin; chicken VAMP-1 and VAMP-2; and Torpedo VAMP-1.Thus, a BoNT/G recognition sequence can correspond, for example, to asegment of human VAMP-1 or VAMP-2, mouse VAMP-1 or VAMP-2, bovine VAMP-1or VAMP-2, rat VAMP-1 or VAMP-2, rat cellubrevin, chicken VAMP-1 orVAMP-2, Torpedo VAMP-1, or another naturally occurring protein sensitiveto cleavage by BoNT/G. Furthermore, as shown in Table H above,comparison of native VAMP amino acid sequences cleaved by BoNT/G revealsthat such sequences are not absolutely conserved (see, also, FIG. 6),indicating that a variety of amino acid substitutions and modificationsrelative to a naturally occurring BoNT/G-sensitive VAMP sequence can betolerated in a BoNT/G substrate useful in the invention.

A variety of TeNT substrates can be useful in the compositions andmethods disclosed herein. A TeNT substrate useful in the invention canhave, for example, at least six consecutive residues of VAMP, the sixconsecutive residues including Gln-Phe, or a peptidomimetic thereof. Asan example, such a TeNT substrate can have at least six consecutiveresidues of human VAMP-2, the six consecutive residues includingGln₇₆-Phe₇₇, or a peptidomimetic thereof. In one embodiment, a TeNTsubstrate contains the amino acid sequenceGly-Ala-Ser-Gln-Phe-Glu-Thr-Ser (SEQ ID NO: 48), or a peptidomimeticthereof. In another embodiment, the TeNT substrate contains residues 33to 94 of human VAMP-2 (SEQ ID NO: 11); residues 25 to 93 of human VAMP-2(SEQ ID NO: 11); or residues 27 to 116 of rat VAMP-2 (SEQ ID NO: 109),or a peptidomimetic of one of these sequences.

As used herein, the term “tetanus toxin recognition sequence” means ascissile bond together with adjacent or non-adjacent recognitionelements sufficient for detectable proteolysis at the scissile bond by atetanus toxin under appropriate conditions. A scissile bond cleaved byTeNT can be, for example, Gln-Phe.

A variety of TeNT recognition sequences are well known in the art or canbe defined by routine methods and include sequences corresponding tosome or all of the hydrophilic core of a VAMP protein such as humanVAMP-1 or human VAMP-2. A TeNT recognition sequence can include, forexample, residues 25 to 93 or residues 33 to 94 of human VAMP-2 (SEQ IDNO: 11; Cornille et al., Eur. J. Biochem. 222:173-181 (1994); Foran etal., Biochem. 33: 15365-15374 (1994)); residues 51 to 93 or residues 1to 86 of rat VAMP-2 (SEQ ID NO: 109; Yamasaki et al., supra, 1994); orresidues 33 to 94 of human VAMP-1 (SEQ ID NO: 10). A TeNT recognitionsequence also can include, for example, residues 25 to 86, residues 33to 86 or residues 51 to 86 of human VAMP-2 (SEQ ID NO: 11) or rat VAMP-2(SEQ ID NO: 109). It is understood that a similar TeNT recognitionsequence can be prepared, if desired, from a corresponding (homologous)segment of another TeNT-sensitive VAMP isoform or species homolog suchas human VAMP-1 or sea urchin or Aplysia VAMP.

Thus, a TeNT recognition sequence can correspond to a segment of aprotein that is sensitive to cleavage by tetanus toxin, or can besubstantially similar to a segment of a TeNT-sensitive protein. As shownin Table H above, a variety of naturally occurring proteins sensitive tocleavage by TeNT are known in the art and include, for example, human,mouse and bovine VAMP-1 and VAMP-2; rat VAMP-2; rat cellubrevin; chickenVAMP-2; Torpedo VAMP-1; sea urchin VAMP; Aplysia VAMP; squid VAMP; C.elegans VAMP; Drosophila n-syb; and leech VAMP. Thus, a TeNT recognitionsequence can correspond, for example, to a segment of human VAMP-1 orVAMP-2, mouse VAMP-1 or VAMP-2, bovine VAMP-1 or VAMP-2, rat VAMP-2, ratcellubrevin, chicken VAMP-2, Torpedo VAMP-1, sea urchin VAMP, AplysiaVAMP, squid VAMP, C. elegans VAMP, Drosophila n-syb, leech VAMP, oranother naturally occurring protein sensitive to cleavage by TeNT.Furthermore, comparison of native VAMP amino acid sequences cleaved byTeNT reveals that such sequences are not absolutely conserved (Table Hand FIG. 6), indicating that a variety of amino acid substitutions andmodifications relative to a naturally occurring TeNT-sensitive VAMPsequence can be tolerated in a TeNT substrate useful in the invention.

A clostridial toxin substrate useful in a substrate composition, cell ormethod of the invention can include one or multiple clostridial toxincleavage sites for the same or different clostridial toxins. Inparticular embodiments, the invention provides a substrate composition,cell or method in which the clostridial toxin substrate contains asingle clostridial toxin cleavage site. In other embodiments, theinvention provides a substrate composition, cell or method in which theclostridial toxin substrate contains multiple cleavage sites for thesame clostridial toxin. These cleavage sites can be accompanied by thesame or different clostridial toxin recognition sequences. As anexample, a substrate composition of the invention can include aclostridial toxin substrate having multiple cleavage sites for the sameclostridial toxin intervening between the same donor fluorophore andacceptor. A clostridial toxin substrate useful in a substratecomposition, cell or method of the invention can contain, for example,two or more, three or more, five or more, or ten or more cleavage sitesfor the same clostridial toxin. A clostridial toxin substrate useful inthe invention also can have, for example, two, three, four, five, six,seven, eight, nine or ten cleavage sites for the same clostridial toxin;the multiple cleavage sites can intervene between the same or differentdonor fluorophore-acceptor pairs.

A clostridial toxin substrate useful in a substrate composition, cell ormethod of the invention also can include cleavage sites and recognitionsequences for different clostridial toxins. In particular embodiments,the invention provides a substrate composition, cell or method in whichthe clostridial toxin substrate includes multiple cleavage sites fordifferent clostridial toxins all intervening between the same donorfluorophore-acceptor pair. A substrate composition, cell or method ofthe invention can include a clostridial toxin substrate having, forexample, cleavage sites for two or more, three or more, or five or moredifferent clostridial toxins all intervening between the same donorfluorophore-acceptor pair. A substrate composition, cell or method ofthe invention also can incorporate a clostridial toxin substrate inwhich, for example, cleavage sites for two or more, three or more, orfive or more different clostridial toxins intervene between at least twodonor fluorophore-acceptor pairs. In particular embodiments, theinvention provides a substrate composition, cell or method including aclostridial toxin substrate having cleavage sites for two, three, four,five, six, seven or eight different clostridial toxins, where thecleavage sites intervene between the same or different donorfluorophore-acceptor pairs. In further embodiments, the inventionprovides a substrate composition, cell or method in which theclostridial toxin substrate has, for example, any combination of two,three, four, five, six, seven or eight cleavage sites for anycombination of the following clostridial toxins: BoNT/A, BoNT/B,BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G and TeNT.

All journal article, reference and patent citations provided above, inparentheses or otherwise, whether previously stated or not, areincorporated herein by reference in their entirety.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

1. A cell comprising (a) at least one receptor that binds a clostridialtoxin, and (b) a clostridial toxin substrate, said clostridial toxinsubstrate comprising: (i) a donor fluorophore; (ii) an acceptor havingan absorbance spectrum overlapping the emission spectrum of said donorfluorophore; and (iii) a clostridial toxin recognition sequencecomprising a clostridial toxin recognition sequence comprising aclostridial toxin P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ cleavage sitesequence, said clostridial toxin P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′cleavage site sequence intervening between said donor fluorophore andsaid acceptor, wherein, under the appropriate conditions, resonanceenergy transfer is exhibited between said donor fluorophore and saidacceptor.
 2. The cell of claim 1, wherein said cell is a neuron.
 3. Thecell of claim 2, wherein said neuron is a peripheral neuron or a centralnervous system neuron.
 4. The cell of claim 2, wherein said neuron is aselected from the group consisting of a primary cell, a cultured cell,an established cell, a normal cell, a transformed cell, a tumor cell, aninfected cell and a transfected cell.
 5. The cell of claim 4, whereinsaid established line is a neuronal cell line selected from the groupconsisting of a neuroblastoma cell line, a hybrid neuronal cell line, amotor neuron cell line, a spinal cord cell line, a cerebral cortex cellline, a dorsal root ganglia cell line and a hippocampal cell line. 6.The cell of claim 1, wherein said cell is a non-neuronal cell.
 7. Thecell of claim 6, wherein said non-neuronal cell is selected from thegroup consisting of a glandular cell, an anterior pituitary cell, anadrenal cell, a pancreatic cell, an epithelial cell, a muscle cell, afibroblast, a neutrophil, an eosinophil, a mast cell, a stomach cell, ahepatocyte, a kidney cell and an ovarian cell.
 8. The cell of claim 6,wherein said non-neuronal cell is a selected from the group consistingof a primary cell, a cultured cell, an established cell, a normal cell,a transformed cell, a tumor cell, an infected cell and a transfectedcell.
 9. The cell of claim 8, wherein said established line is anon-neuronal cell line selected from the group consisting of achromaffin cell line, an enterochromaffin cell line, a pancreatic isletβ cell line, a pancreatic acinar cell line, an insulinoma HIT cell line,an INS-1 cell line, a steroid-producing ovarian cell line, an innermedullary collecting duct (IMCD) cell line, a platelet cell line, aneutrophil cell line, an eosinophil cell line, a mast cell line and aglucose transporter translocation cell line.
 10. The cell of claim 1,wherein said receptor is selected from the group consisting of a highaffinity receptor, a low affinity receptor, an endogenous receptor andan exogenous receptor.
 11. The cell of claim 1, wherein said donorfluorophore is selected from the group consisting of blue fluorescentprotein, cyan fluorescent protein, green fluorescent protein, yellowfluorescent protein and red fluorescent protein.
 12. The cell of claim1, wherein said acceptor is a fluorophore selected from the groupconsisting of blue fluorescent protein, cyan fluorescent protein, greenfluorescent protein, yellow fluorescent protein and red fluorescentprotein.
 13. The cell of claim 1, wherein said acceptor is anon-fluorescent molecule.
 14. The cell of claim 1, wherein saidclostridial toxin substrate is a botulinum toxin substrate, wherein saidclostridial toxin P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognitionsequence is a botulinum toxin recognition sequence comprising abotulinum toxin P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ cleavage sitesequence.
 15. The cell of claim 14, wherein said botulinum toxinsubstrate is a BoNT/A substrate, wherein said botulinum toxinP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence is a BoNT/Arecoginition sequence comprising a BoNT/AP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ cleavage site sequence.
 16. The cellof claim 15, wherein said BoNT/A P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′cleavage site comprises at least six consecutive residues of SNAP-25 ora peptidomimetic thereof, said six consecutive residues comprisingGln-Arg, or a peptidomimetic thereof.
 17. The cell of claim 16, whereinsaid BoNT/A P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ cleavage site sequencecomprises SEQ ID NO:
 41. 18. The cell of claim 14, wherein saidbotulinum toxin substrate is a BoNT/B substrate, wherein said botulinumtoxin P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence is aBoNT/B recognition sequence comprising a BoNT/BP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ cleavage site sequence.
 19. The cellof claim 18, wherein said BoNT/B P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′cleavage site sequence comprises at least six consecutive residues ofVAMP, or a peptidomimetic thereof, said six consecutive residuescomprising Gln-Phe, or a peptidomimetic thereof.
 20. The cell of claim19, wherein said BoNT/B P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ cleavage siteregion sequence comprises SEQ ID NO:
 42. 21. The cell of claim 14,wherein said botulinum toxin substrate is a BoNT/C1 substrate, whereinsaid botulinum toxin P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognitionsequence is a BoNT/C1 recognition sequence comprising a BoNT/C1P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ cleavage site sequence.
 22. The cellof claim 21, wherein said BoNT/C1 P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′cleavage site sequence comprises at least six consecutive residues ofsyntaxin, or a peptidomimetic thereof, said six consecutive residuescomprising Lys-Ala, or a peptidomimetic thereof.
 23. The cell of claim21, wherein said BoNT/C1 P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ cleavagesite sequence comprises at least six consecutive residues of SNAP-25, ora peptidomimetic thereof, said six consecutive residues comprisingArg-Ala, or a peptidomimetic thereof.
 24. The cell of claim 23, whereinsaid BoNT/C1 P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ cleavage site sequencecomprises SEQ ID NO:
 43. 25. The cell of claim 14, wherein saidbotulinum toxin substrate is a BoNT/D substrate, wherein said botulinumtoxin P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence is aBoNT/D recognition sequence comprising a BoNT/DP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ cleavage site sequence.
 26. The cellof claim 25, wherein said BoNT/D P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′cleavage site sequence comprises at least six consecutive residues ofVAMP, or a peptidomimetic thereof, said six consecutive residuescomprising Lys-Leu, or a peptidomimetic thereof.
 27. The cell of claim26, wherein said BoNT/D P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ cleavage sitesequence comprises SEQ ID NO:
 44. 28. The cell of claim 14, wherein saidbotulinum toxin substrate is a BoNT/E substrate, wherein said botulinumtoxin P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence is aBoNT/E recognition sequence comprising a BoNT/EP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ cleavage site sequence.
 29. The cellof claim 28, wherein said BoNT/E P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′cleavage site sequence comprises at least six consecutive residues ofSNAP 25, or a peptidomimetic thereof, said six consecutive residuescomprising Arg-Ile, or a peptidomimetic thereof.
 30. The cell of claim29, wherein said BoNT/E P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ cleavage sitesequence comprises SEQ ID NO:
 45. 31. The cell of claim 14, wherein saidbotulinum toxin substrate is a BoNT/F substrate, wherein said botulinumtoxin P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence is aBoNT/F recognition sequence comprising a BoNT/FP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ cleavage site sequence.
 32. The cellof claim 31, wherein said BoNT/F P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′cleavage site sequence comprises at least six consecutive residues ofVAMP, or a peptidomimetic thereof, said six consecutive residuescomprising Gln-Lys, or a peptidomimetic thereof.
 33. The cell of claim32, wherein said BoNT/F P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ cleavage sitesequence comprises SEQ ID NO:
 46. 34. The cell of claim 14, wherein saidbotulinum toxin substrate is a BoNT/G substrate, wherein said botulinumtoxin P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence is aBoNT/G recognition sequence comprising a BoNT/GP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ cleavage site sequence.
 35. The cellof claim 34, wherein said BoNT/G P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′cleavage site sequence comprises at least six consecutive residues ofVAMP, or a peptidomimetic thereof, said six consecutive residuescomprising Ala-Ala, or a peptidomimetic thereof.
 36. The cell of claim35, wherein said BoNT/G P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ cleavage sitesequence comprises SEQ ID NO:
 47. 37. The cell of claim 1, wherein saidclostridial toxin substrate is a TeNT toxin substrate, wherein saidclostridial toxin P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognitionsequence is a TeNT recognition sequence comprising a TeNTP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ cleavage site sequence.
 38. The cellof claim 37, wherein said TeNT P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′cleavage site sequence comprises at least six consecutive residues ofVAMP, or a peptidomimetic thereof, said six consecutive residuescomprising Gln-Phe, or a peptidomimetic thereof.
 39. The cell of claim38, wherein said TeNT P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ cleavage sitesequence comprises SEQ ID NO:
 48. 40. The cell of claim 1, wherein saidclostridial toxin substrate comprises only one clostridial toxinP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence.
 41. The cell ofclaim 1, wherein said clostridial toxin substrate comprises multipleclostridial toxin P₅-P₄-P₃-P₂-P₁-P₁-P₂′-P₃′-P₄′-P₅′ recognitionsequences from the same clostridial toxin.
 42. The cell of claim 1,wherein said clostridial toxin substrate comprises multiple clostridialtoxin P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequences fromdifferent clostridial toxins.
 43. The cell of claim 42, wherein saidmultiple clostridial toxin P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′recognition sequences comprise a BoNT/AP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence.
 44. The cell ofclaim 42, wherein said multiple clostridial toxinP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequences comprise aBoNT/A P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence and aBoNT/C1 P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence.
 45. Thecell of claim 42, wherein said multiple clostridial toxinP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequences comprise aBoNT/A P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence, aBoNT/C1 P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence and aBoNT/E P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence.
 46. Thecell of claim 42, wherein said multiple clostridial toxinP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequences comprise aBoNT/B P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence.
 47. Thecell of claim 42, wherein said multiple clostridial toxinP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequences comprise aBoNT/B P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence and aBoNT/G P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence.
 48. Thecell of claim 42, wherein said multiple clostridial toxinP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequences comprise aBoNT/D P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence.
 49. Thecell of claim 42, wherein said multiple clostridial toxinP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequences comprise aBoNT/D P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence and aBoNT/F P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence.
 50. Thecell of claim 42, wherein said multiple clostridial toxinP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequences comprise aBoNT/B P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence, a BoNT/DP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence, a BoNT/FP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence and a BoNT/GP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence.
 51. The cell ofclaim 42, wherein said multiple clostridial toxinP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequences comprise aBoNT/A P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence and aBoNT/B P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ recognition sequence.
 52. Thecell of claim 1, wherein said donor fluorophore and said acceptor areseparated by at most 70 residues.
 53. The cell of claim 1, wherein saiddonor fluorophore and said acceptor are separated by at most 100residues.
 54. The cell of claim 1, wherein said donor fluorophore andsaid acceptor are separated by at most 150 residues.
 55. The cell ofclaim 1, wherein said donor fluorophore and said acceptor are separatedby at most 200 residues.
 56. The cell of claim 1, wherein said donorfluorophore and said acceptor are separated by the full length of anaturally-occurring clostridial toxin target protein.
 57. The cell ofclaim 1, wherein said clostridial toxin substrate of any one of claims1, 11-56 is transiently transfected into said cell.
 58. The cell ofclaim 1, wherein said clostridial toxin substrate of any one of claims1, 11-56 is encoded by a nucleic acid molecule.
 59. The cell of claim58, wherein said nucleic acid molecule is linked to a regulatory elementselected from the group consisting of a constitutive regulatory elementand an inducible regulatory element.
 60. The cell of either claim 58 or59, wherein said nucleic acid molecule is transiently transfected intosaid cell.
 61. The cell of either claim 58 or 59, wherein said nucleicacid molecule is stably transfected into said cell.